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From the Department of Biochemistry & Biophysics
Stockholm University, Stockholm, Sweden
STUDIES ON THE CONFORMATION
OF TRANSMEMBRANE POLYPEPTIDES
IN MEMBRANE PROTEINS
MARIKA CASSEL
Stockholm 2005
2
Marika Cassel
Intellecta Docusys AB, Stockholm 2005
ISBN 91-7155-175-1
3
Till Tobias
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Marika Cassel
Abstract
The major aim of the studies that this thesis is based on has been to better define the
topological determinants of the formation of so-called helical hairpins during
membrane protein assembly in the ER membrane.
The helical hairpin is a basic folding unit in membrane proteins. It is composed of
two closely spaced transmembrane helices with a short connecting loop and it is
believed to be inserted into the membrane as one compact unit. It is becoming
increasingly clear that the helical hairpin is a very common structural element in
membrane proteins and a detailed understanding of its properties is of central
importance.
We demonstrate that the efficiency of formation of helical hairpins depends both on
the overall length of the hydrophobic segment, on the amino acids flanking the
transmembrane segment, and on the identity of the central, potentially turn-forming
residues. We also show that interhelical hydrogen bonds between pairs of Asn or
Asp residues can induce helical hairpin formation.
A detailed topology mapping is also reported for the Escherichia coli inner membrane
chloride channel YadQ, a protein for which the X-ray structure is known. Our results
provide a critical test of the reporter fusion approach and offer new insights into the
YadQ folding pathway.
In summary, the results present in this thesis have increased our understanding of
the determinants of membrane protein topology and structure. Furthermore, the
information obtained can be used to improve current models for predictions of
membrane protein topology.
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Main References
This thesis is based on the following original papers that are reprinted in the second
part of the thesis. In the text, these papers will be referred to by their roman
numerals.
Paper I: Monné, M. Hermansson, M., von Heijne, G. (1999) A turn propensity scale
for transmembrane helices. Journal of Molecular Biology 288:141-145
Paper II: Sääf, A. Hermansson, M., von Heijne, G. (2000) Formation of cytoplasmic
turns between two closely spaced transmembrane helices during membrane protein
integration into the ER membrane. Journal of Molecular Biology 301:191-197
Paper III: Hermansson, M., Monné, M., von Heijne, G. (2001) Formation of helical
hairpins during membrane protein integration into the endoplasmic reticulum
membrane. Role of the N and C-terminal flanking regions. Journal of Molecular Biology
314:1171-1179
Paper IV: Hermansson, M., von Heijne, G. (2003) Inter-helical hydrogen bond
formation during membrane protein integration into the ER membrane. Journal of
Molecular Biology 334:803-809
Paper V: Cassel, M., von Heijne, G. (2005) Confronting fusion protein-based
membrane protein topology mapping with reality: the Escherichia coli ClC chloride
channel. Manuscript
The published articles have been reprinted with permission from Elsevier Ltd
(Papers I-IV)
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Marika Cassel
Table of contents
Abstract…………………………………………………………………………………………………………………………… 4
Main References………………………………………………………………………………………………………… 5
Abbreviations……………………………………………………………………………………………………………… 8
Introduction………………………………………………………………………………………………………………….. 9
Biological membranes……………………………………………………………………………………………... 9
Membrane proteins………………………………………………………………………………………………….. 10
Peripheral membrane proteins…………………………………………………………………………… 10
Lipid-linked membrane proteins………………………………………………………………………... 10
Facultative membrane proteins…………………………………………………………………………..11
Integral membrane proteins………………………………………………………………………………. 11
Membrane protein structure…………………………………………………………………………………... 12
α-helical membrane proteins……………………………………………………………………………... 13
β-barrel membrane proteins ……………………………………………………………………………… 14
Experimental structure determination of membrane proteins…………………………. 14
Determination of 3D-structure…………………………………………………………………………. 14
Studies of dynamic properties……………………………………………………………………………. 15
Computer simulations……………………………………………………………………………………….. 16
Membrane protein topology…………………………………………………………………………………... 16
Experimental topology determination……………………………………………………………….. 16
Protease-protection assay……………………………………………………………………... 17
Cystein scanning……………………...……………………………………………………………...17
Gene fusion analysis……………………………………………………………………………… 17
Mapping of loop regions………………………………………………………………………. 18
Computerized topology determination……………………………………………………………… 19
Topogenic determinants……………………………………………………………………………………. 20
Topogenic signals………………………………………………………………………………….. 20
Single-spanning membrane proteins…………………………………………………. 20
Charged residues…………………………………………………………………………………… 21
Multi-spanning membrane proteins…………………………………………………… 22
Biosynthesis and targeting to the ER membrane………………………………………………… 23
The protein-conducting translocon………………………………………………………………………. 24
Translocation……………………………………………………………………………………………………... 26
Translocon-mediated membrane insertion………………………………………………………… 26
Folding of extramembraneous domains………………………………………………………………..27
Helix-helix packing in the membrane…………………………………………………………………... 28
Comments on Methodology………………………………………………………………………………….. 29
Model system used to study helical hairpin formation………………………………………29
Model protein – Leader peptidase……………………………………………………………………… 29
Manipulation of the model protein……………………………………………………………………. 30
Glycosylation mapping……………………………………………………………………………………… 30
Alkaline extraction……………………………………………………………………………………………. 31
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Alkaline phosphatase as a topology reporter protein…………………..……………………. 31
Green fluorescent protein as a topology reporter protein…………...…………………….. 31
Results and Discussion……………………………………………………………………………………………. 32
Paper I……………………………………………………………………………………………………………………….. 32
Paper II………………………………………………………………………………………………………………………. 33
Paper III…………………………………………………………………………………………………………………….. 34
Paper IV…………………………………………………………………………………………………………………….. 35
Paper V……………………………………………………………………………………………………………………… 36
Concluding Remarks………………………………………………………………………………………………… 38
Acknowledgements…………………………………………………………………………………………………… 39
References……………………………………………………………………………………………………………………… 41
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Marika Cassel
Abbreviations
AFM
ATP
CD
cryo-EM
EM
ER
FA
FTIR
GFP
HC
IF
IMP
kDa
MC
MD
MP
NMR
OST
PMP
PKA
rSA
SA
SP
SPase
ST
SPC
SRP
TM
TRAM
TRAP
TROSY
Atomic force microscopy
Adenosine triphosphate
Circular dichroism
Cryo-electron microscopy
Electron microscopy
Endoplasmic reticulum
Fatty acid
Fourier transform infrared
Green fluorescent protein
Hydrocarbon core
Interfacial regions
Integral membrane protein
kilo Dalton
Monte Carlo
Molecular dynamics
Membrane protein
Nuclear magnetic resonance
Oligosaccharyl transferase
Peripheral membrane protein
Protein kinase A
Reverse signal anchor
Signal anchor
Signal peptide
Signal peptidase complex
Stop-transfer
Signal peptidase complex
Signal recognition particle
Transmembrane
Translocating chain associated membrane protein
Translocon-associated protein complex
Transverse optimized NMR spectroscopy
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Introduction
Biological membranes
All living cells are enclosed by a membrane (the plasma membrane) which
separates the cell from the surrounding environment. Biological membranes also
form boundaries within the eukaryotic cell where they divide the internal space
in the cell into distinct compartments, so-called organelles. Each organelle, such
as endoplasmic reticulum (ER), golgi network, lysosomes, peroxisomes, nucleus,
mitochondria, chloroplasts, etc. contains a specific complement of proteins and
other molecules and has a unique role in the cell. Besides acting as selectively
permeable barriers, membranes are involved in thousands of essential and
diverse functions in the cell, such as transport of nutrients, adenosine
triphosphate (ATP) synthesis, ion conduction, signal transduction, cell-cell
interactions, respiration, development, vision, hearing, fertilization and many
more.
Cellular membranes are highly organized structures, built from two basic
components, lipids and membrane proteins. The fluid mosaic model describes the
organisation and features common to all membranes and predicts free rotational
and lateral diffusion of proteins and lipids within the plane of the membrane,
resulting in their random distribution (Singer and Nicolson, 1972). However, to
account for lateral heterogeneities in membranes, which is a result of proteinprotein, protein–lipid and lipid-lipid interactions, this model has recently been
refined (Vereb et al., 2003).
Membrane lipids are divided into three classes: glycerophospholipids,
sphingolipids and cholesterol, where glycerophospholipids are the main lipid
constituent of most biomembranes. Lipids are amphiphilic molecules, i.e. they
contain both hydrophobic and hydrophilic regions. In glycerophospholipids and
sphingolipids, the hydrophobic part is composed of the fatty acid (FA)
hydrocarbon chains, which usually are unbranched and have an even number of
carbon atoms, usually 16 or 18. The FA chains are either saturated with hydrogen
atoms or have one or more unsaturated double bonds. The polar headgroups of
the lipids have different charges and are hydrophilic. In cholesterol the whole
molecule except the hydroxyl group on carbon-3 is hydrophobic.
Membrane lipids interact noncovalently with each other and are packed side by
side forming the “lipid bilayer”, in which the hydrophobic FA chains are oriented
towards the interior of the membrane, forming a 30Å wide hydrophobic
hydrocarbon core (HC). The hydrophilic lipid headgroups are on the surfaces of
the membrane core, forming two polar interfacial regions that are 10-15Å wide.
The two layers of lipids in the bilayer are termed the inner and outer leaflet.
Embedded in the lipid bilayer are membrane proteins of various shapes and
10 Marika Cassel
traits. These proteins are either integrated into the membrane - integral
membrane proteins - or peripherally associated with the membrane - peripheral
membrane proteins. Many membrane proteins and lipids contain covalently
attached oligosaccharides. The weak noncovalent interactions between
neighbouring molecules give rise to the membrane fluidity, i.e. the lipids can
move easily within the plane of the membrane. The fluidity of the membrane is
affected by the length of the FA chains and degree of unsaturation in the FA
chains. Maintenance of the bilayer fluidity appears to be essential for normal cell
function.
The membrane surrounding each type of organelle has organelle-specific lipid
and protein composition, which contributes to their unique properties. For
example, the ER membrane is composed mainly of the glycerophospholipid
phosphatidylcholin and to a lesser extent of phosphatidylethanolamine,
phophatidylserin and phosphatidylinositol, while levels of cholesterol and
sphingolipids are low (van Meer, 1989).
The relative proportions of membrane proteins and lipids vary with the type of
membrane. Lipids and membrane proteins are asymmetrically distributed
between the inner and outer leaflet and can also be non-randomly distributed
within a single leaflet, which is seen in membrane microdomains termed lipid
rafts. (Simons and Ikonen, 1997; Simons and Ikonen, 2000). Lipid rafts appear to
be enriched in sphingolipids and cholesterol that form a highly ordered structure
distinct from the surrounding sea of lipids. In addition, specific types of
membrane proteins implicated in signal transduction, membrane trafficking
within cells and membrane protein sorting, have been shown to be localized to
lipid rafts (Laude and Prior, 2004).
Membrane proteins
Peripheral membrane proteins
Membrane proteins are classified according to how tightly they are associated to
the membrane. Peripheral membrane proteins are loosely associated to the
membrane by hydrogen bonding and electrostatic interactions and do not interact
strongly with the hydrophobic interior of the bilayer. Instead they interact with
hydrophilic domains of integral membrane proteins in the membrane and with
the polar headgroups of membrane lipids. Some peripheral membrane proteins
serve as regulators of membrane spanning enzymes, while others interact with
the cytoskeleton and maintain or alter the cellular shape. Structurally, peripheral
membrane proteins are similar to globular proteins.
Lipid-linked membrane proteins
Another class of membrane proteins is the lipid-linked membrane proteins. These
proteins have one or more covalently attached lipid that anchors the protein to
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the lipid bilayer. The anchoring lipids may be of several types: long-chain fatty
acids, isoprenoids or sterols. Lipid-protein assemblies containing these three
types of lipids are found in the inner leaflet of the plasma membrane, while lipidprotein assemblies containing glycosylated derivatives of phosphatidylinositol
are found only in the outer leaflet of the plasma membrane.
Facultative membrane proteins
In some microorganisms intricate mechanisms have evolved where secreted
proteins or insertion peptides, that are soluble in the aqueous phase (so called
pore-forming toxins) partition spontaneously into the host cell membrane where
they fold and/or assemble and form integral membrane protein pores that
promote translocation of toxins into the cytoplasm of the host cell (Krantz et al.,
2005). The membrane spanning part of these pores can either have α-helix
conformation or form β-barrels (Gouaux, 1997; Song et al., 1996; Stroud, 1995).
Integral membrane proteins
Approximately 20-30% of all genes in the fully sequenced genome are predicted
to code for integral membrane proteins (von Heijne, 1996). They are found in all
cellular membranes and carry out a wide range of central functions in the cell.
Some act as signal receptors that transduce signals across the membrane, while
others catalyze specific transport of nutrients, metabolites and ions across the
membrane barriers. In addition, some integral membrane proteins couple the
flow of electrons to the synthesis of ATP and specific transmembrane (TM)
proteins in the plasma membrane are involved in cell-cell interactions, where they
serve as highly active mediators between the cell and its environment or the
interior of an organelle and the cytosol. Many membrane proteins play important
roles in tumor development and in the immune response. Since integral
membrane proteins fulfil many critical functions in the cell, they are of major
medical importance and thus provide interesting targets for the pharmaceutical
industry.
Integral membrane proteins are embedded in the membrane bilayer, in which
they fold and function. They are composed of one or more hydrophobic
membrane spanning segments, linked by hydrophilic regions of the polypeptide
chain, which are exposed either on the extracellular or cytosolic side of the
membrane. The TM segments consist mainly of amino acid residues with
hydrophobic side-chains that protrude outwards and interact with the FA chains
in the lipid bilayer. These hydrophobic protein-lipid interactions tightly anchor
the integral membrane proteins to the hydrophobic core of the membrane.
The hydrophobic environment of the lipid bilayer strongly limits the range of
possible structures for the membrane spanning part of integral membrane
proteins. When a polypeptide chain is surrounded by lipids, the polar peptide
bonds in the backbone are driven to form hydrogen bonds with one another since
there are no water molecules in the lipid environment. Hydrogen bonding
12 Marika Cassel
between peptide bonds is maximized if the polypeptide chain forms a regular αhelix as it crosses the lipid bilayer. Thus, the polypeptide chain of most TM
proteins is thought to cross the lipid bilayer in an α-helical conformation
(Deisenhofer et al., 1985; Henderson et al., 1990; Henderson and Unwin, 1975). An
alternative way for the peptide bonds in the lipid bilayer to satisfy the hydrogenbonding requirements is for multiple strands of polypeptide chains to be
arranged as a β-sheet, in the form of a closed barrel. Not much is known how βbarrel proteins fold, except that a good part of the structure must be present
before the hydrophobic external surface is well defined. Thus, the β-barrel
probably forms before or simultaneously with membrane insertion (Tamm et al.,
2001).
A number of studies suggest that helix bundle proteins fold according to a twostep mechanism. In a first step, individually stable helices are formed and
inserted into the membrane. Subsequently in a second step, these helices interact,
giving rise to tertiary and quaternary structures and thus to a functional TM
structure (Popot and Engelman, 1990). In a membrane the hydrophobic effect
contributes only to the formation and insertion of TM helices (step 1). The
interactions that drive bundle formation (step 2) are not clearly understood. The
formation of salt bridges between oppositely charged residues may be one factor
and the burial of polar amino acids in the protein interior may be another (Bowie,
2000; Zhou et al., 2000). Tight packing between TM helices is important for
protein stability. Theoretical calculations indicate that van der Waals interactions
between apolar TM helices may be enough to drive the formation of helix bundles
in a lipid environment (Bowie, 1997; von Heijne, 1994).
Membrane protein structure
Although many thousands of membrane protein sequences are known, progress
in the structural analysis of these proteins has been hampered by difficulties in
their purification and crystallization. A comprehensive understanding of
fundamental membrane-associated biological processes such as energy
conversion, transport and signal transduction cannot be attained unless the highresolution structures of the membrane proteins involved are known.
To date, only about 100 high-resolution structures of membrane proteins have
been solved [http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html], compared to
about 30,000 globular proteins. Among these around 100 known structures, only
two basic structural features have been found: the α-helical conformation and the
β-barrel fold (Figure 1) (Cowan and Rosenbusch, 1994). Integral membrane
proteins are usually divided into two major classes, each one containing one of
these two structural features (von Heijne, 1994).
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α-helical membrane proteins
The membrane proteins that belong to the so-called helix bundle class, have
membrane domains composed of two or more TM α-helices usually consisting of
a stretch of about 15-30 amino acids with a central hydrophobic domain flanked
by more polar residues (Wallin et al., 1997). The loops connecting two TM αhelices can be as short as three residues (Bowie, 1997).
The hydrophobic domain of TM α-helices is composed of mainly, but not entirely,
of hydrophobic amino acid residues (Ile, Leu, Val, Ala, Phe and Gly ). The TMs
may include a limited number of polar or potentially charged groups; Cys, Thr
and Ser residues often form H-bonds bonds to the main chain. Aromatic side
chains, particularly Tyr and Trp, are often located near the boundary between the
hydrophilic and hydrophobic regions of the lipid bilayer, both in helical and βbarrel membrane proteins found within the lipid head group region and can
serve as membrane anchors (Schiffer et al., 1992). Pro and Gly are found more
commonly in TM α-helices than in soluble protein helices. Pro is sometimes
found near the middle of TM α-helices in multispanning membrane proteins
(Jones et al., 1994) where it can induce a bend in the helix. Such Pro residues,
which tend to be highly conserved, result in a kink and disrupt several helical
hydrogen bonds (von Heijne, 1991).
Figure 1 . Two membrane proteins containing different basic structural features. A helix bundle
protein, bacteriorhodopsin (Belrahli et al, 1999), (left) and a β-barrel protein, maltoporin (Schirmer et
al., 1995), (right).
The TM α-helices are oriented more or less perpendicular to the plane of the
membrane with somewhat variable tilts. Helix bundle membrane proteins are
found in almost all membranes except in the bacterial outer membrane. One
example of a helix-bundle protein is bacteriorhodopsin, a light-driven proton
pump from Halobacterium halobium. It contains seven TM α-helices and was the
first integral membrane protein to be shown to consist of a helix bundle. Electron
microscopy of two-dimensional crystals of this protein was used to derive the
structure (Henderson and Unwin, 1975). Such 2D-crystals or ordered sheets of
membrane proteins are often easier to obtain than 3D-crystals, and in some cases
14 Marika Cassel
they occur in vivo, in the plane of the membrane (Brändén and Tooze, 1991).
Bacteriorhodopsin is one of the best structurally and functionally characterized
integral membrane proteins.
β-barrel membrane proteins
The other class of integral membrane protein is the so-called β-barrel class. In
proteins containing this structural feature, the membrane-embedded domain is
composed of a large, antiparallel β-sheet barrel. These proteins are found in the
outer membrane of Gram-negative bacteria and in the mitochondrial outer
membrane (Henderson et al., 1990; Henderson and Unwin, 1975). An example of
typical β-barrel proteins in bacteria is the porins. The porins form rather large
pores through the membrane, through which specific small substrates can diffuse
passively (Buchanan, 1999; Schulz, 2000). In these structures, every second amino
acid residue in each strand is hydrophobic and faces out towards the lipid
environment, while the surface facing the channel is usually more hydrophilic
(Cowan et al., 1992; Weiss et al., 1991).
Experimental structure determination of membrane proteins
Integral membrane proteins have not been studied as thoroughly as globular
proteins, for simple technical reasons. Since membrane proteins have both
hydrophobic and hydrophilic regions on their surfaces, they are not soluble in
aqueous solution and denature in organic solvents. Because of these amphipatic
properties, they are tricky to handle biochemically. In addition, membrane
proteins are hard to overexpress and they seldom yield high-quality 2D or 3D
crystals.
Determination of 3D structures
There are three techniques that can be used for determining the 3D structure of
protein molecules; X-ray crystallography and nuclear magnetic resonance (NMR),
which have yielded by far the largest numbers of protein structures to date, but
also electron crystallography (von Heijne, 1996). Each technique has its strengths
and weaknesses but they all require large amounts of very pure protein, which
often is the bottleneck for membrane proteins because of the difficulties with
overexpression, purification and production of 3D-crystals. However, the
difficulties in crystallizing membrane proteins are beginning to be overcome and
X-ray structures are now available for at least one example of most kinds of
transporters, sensors and G-protein coupled receptors.
Several different NMR techniques are used for structural studies on membrane
proteins. Solution NMR can be used in order to obtain information about
membrane proteins in detergent micelles that closely mimic the natural
membrane environment. This technique is carried out on macromolecules in
solution and crystals are not needed. Solution NMR is generally considered to be
15
restricted to proteins up to 30-50kDa, but recently significantly larger proteins
have been studied (Fiaux et al 2000). This technique is preferably used on proteins
with β-barrel fold. Recently, larger β-barrel outer membrane proteins such as
OmpX (Fernandez et al., 2001; Fernandez et al., 2002), OmpA (Arora et al., 2001;
MacKenzie et al., 1997; Tamm et al., 2003) and PagP (Hwang et al., 2002) have
been studied using transverse optimized NMR spectroscopy (TROSY) (Pervushin
et al., 1997). Solid-state NMR can be applied to membrane proteins in native
membranes or in phospholipid bilayers and can reveal the peptide backbone
structure and intramolecular distances (Opella, 1997).
Conventionally, methods for solubilization, purification and subsequent
crystallization of membrane proteins have employed detergents. However, a
novel non-detergent concept has been developed recently, using highly viscous,
optically transparent, lipidic cubic phases. The materials used are composed of
very high amounts of hydrated lipids (Landau and Rosenbusch, 1996) and several
integral membrane proteins, from one single mesophase system based on
monoolein, have been crystallized (Chiu et al., 2000). This technique provides
new possibilities to generate well-ordered 3D crystals of membrane proteins, but
it is so far only applicable to coloured membrane proteins, since the formation of
uncoloured membrane protein crystals cannot easily be detected in the lipidic
cubic phase.
Cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) can also
be used to obtain structural information. These techniques have been used to
determine structures of bacteriorhodopsin (Subramaniam and Henderson, 1999),
aquaporins (Murata et al., 2000; Scheuring et al., 2002) and Na+, K+-ATPase with
resolutions in the order of 3.2-9.2Å.
Studies of dynamic properties
Although structurally ordered, many membrane proteins are flexible and
dynamic molecules and must go through conformational changes in order to
perform their functions. Our knowledge of these changes is extremely limited,
because of the small number of known membrane protein structures available
today. In addition, these high-resolution structures only provide snapshots of
single conformations and reveal only one aspect of the behaviour, unless
structures of all the intermediates of the conformational rearrangements are
available. This is the case of bacteriorhodopsin (Lanyi, 2004) where structural
models for all the intermediates in the photochemical cycle are available. This
provides a clear picture of the conformational rearrangements resulting from
photoisomerization of the retinal chromophore. To obtain information about the
dynamic properties of membrane proteins, including conformational changes,
protein folding and interactions with other molecules, several methods can be
utilized. These methods include for example, circular dichroism (CD)
spectroscopy, polarized Fourier transform infrared (FTIR) spectroscopy and
fluorescence emission spectroscopy (Hunt et al., 1993). NMR can also illuminate
16 Marika Cassel
the dynamic side of membrane protein structure. Conformational dynamics in Ptype ATPases have also been studied under physiological conditions using
voltage clamp fluorometry.
Computer simulations
Computer simulations can complement biophysical experimental approaches to
examine membrane protein structures, by extrapolating from static high–
resolution structures. In this way, the dynamics of TM peptides can be modelled.
Monte Carlo (MC) and molecular dynamics (MD) simulations are used to study
membrane proteins structure, dynamics and energetics. Folding kinetics of the
first two TM α-helices of bacteriorhodopsin have been studied using computer
simulations, which show that the helices form rapidly. However, before the right
packing in the membrane is found, a long period of consolidation follows
(Orlandini et al., 2000).
Membrane protein topology
For membrane proteins with unknown three-dimensional structure, it is
necessary to use alternative approaches to obtain structural information. By using
computerized sequence-based prediction methods or different experimental
approaches, it is possible to produce quite reliable topology models for
membrane proteins. With such models, it is possible to describe the secondary
structure of the membrane embedded domain, the number of peptide segments
that traverse the membrane and their respective orientation in the membrane. For
most membrane proteins, as a result of the reasons mentioned above, the
topology model is the only structural information available. Although it is not as
informative as a three-dimensional structure, it still provides important
information. This was shown in a recent study where a global topology analysis
of the Escherichia coli inner membrane proteome made it possible to categorize
proteins according to their different topologies and functions (Daley et al., 2005).
Only a small number of membrane proteins are known to adopt two or more
distinct topological orientations (Levy, 1996; Ma and Chang, 2004; UbarretxenaBelandia et al., 2003; Ubarretxena-Belandia and Tate, 2004). Examples of
homologous proteins with opposite orientations in the membrane have been
reported (Sääf et al., 1999).
Experimental topology determination
Several different biochemical and molecular biology techniques are available for
topology determination. In these methods, enzymes or other reagents are
involved, which are active on, or have access to, only one side of the membrane
(Traxler et al., 1993). Here, some of the most frequently used methods available
for topology determination are summarized.
17
Protease-protection assay
The protease-protection assay can be used to confirm the topology of a membrane
protein and to get information about the localization of membrane protein
domains. The proteases trypsin and proteinase K can be added to microsomes or
sphaeroplasts, containing the membrane protein under study. These enzymes
degrade the exposed domains, while unexposed cytoplasmic and luminal
domains remain undegraded. The remaining, protease-protected part of the
membrane protein can be detected by SDS-PAGE .
Cysteine scanning
The cysteine scanning method also provides an excellent tool for determining the
localization of intra- and extracellular domains of membrane proteins. In this
approach, the amino acid cysteine with its reactive sulfhydryl group is
introduced at a specific site of choice, after all naturally occuring cysteine residues
have been removed from the target protein (Möller and Rydström, 1999). The
sulfhydryl group can be modified by special reagents with different chemical
properties. A shift in the appearent molecular weight of the modified protein can
be detected by SDS-PAGE analysis, but alternative assays can also be used for
detecting the cystein residue exposure to the reagent. The cysteine scanning
method has been used in combination with biophysical approaches to study e.g.,
helix packing in LacY in Escherichia coli (Kaback et al., 1997).
Gene fusion analysis
The most widely used method for membrane protein topology determination is
gene fusion analysis (Ikeda et al., 2003; Möller et al., 2000). In this approach,
extramembraneous domains of the membrane protein under study are fused to a
reporter protein that is active on only one side of the membrane. Most reporter
proteins are enzymes that catalyse a reaction, which can easily be detected by e.g.
spectrophotometric measurements. From the reporter activity it can be
determined whether the reporter-fused domain of the membrane protein is
translocated to the periplasm or if it is retained in the cytoplasm, and a topology
model can be produced.
Usually, 5´-parts of the gene coding for the membrane protein under study is
fused to a reporter gene in a cloning vector (Boyd et al., 1993). The reporter
should only be fused to extramembraneous parts of the membrane protein, to
avoid ambiguous results. To derive a topology model of a polytopic membrane
protein, the reporter has to be fused to each loop region. This is achieved by
successively truncating the full-length gene at the 3´end and fusing it to the
reporter gene.
To produce a reliable topology model it is appropriate to choose two reporter
proteins which have opposite activity profiles and thus can give complementary
results. As seen in paper V, the topological information obtained from the green
18 Marika Cassel
fluorescent protein (GFP) assays, complements the results from the alkaline
phosphatase (PhoA) assays.
The classic example of a reporter protein is PhoA, which has been widely used for
identifying periplasmic loops of membrane proteins expressed in Escherichia coli
(Manoil et al., 1990). PhoA has to form two disulfide bonds in order to become
active and these two bonds can only be formed in the oxidizing environment of
the periplasm (Derman and Beckwith, 1991). When a particular substrate is added
to a bacterial culture expressing the fusion construct, a chromogenic product is
formed if the PhoA domain is in the periplasm, and the PhoA activity can be
quantified spectrophotometrically (Manoil, 1991; Manoil and Beckwith, 1986).
When PhoA is located in the cytoplasm it is unfolded and therefore inactive.
PhoA is highly resistant to proteases when located in the periplasm, but not when
located in the cytoplasm (Roberts and Chlebowski, 1984). This feature can be used
to further confirm the topology.
Another widely used periplasmic reporter protein is β-lactamase (Broome-Smith
and Spratt, 1986; Broome-Smith et al., 1990; Prinz and Beckwith, 1994; Tadayyon
et al., 1992). This bacterial enzyme hydrolyses and inactivates β-lactam antibiotics
such as ampicillin. β-lactamase has a high enzymatic activity when in the
periplasm. Growth of bacterial colonies on an agar plate containing ampicillin is
thus indicative of a periplasmic location of the reporter protein.
β-galactosidase (LacZ) (Froshauer et al., 1988) is another example of a bacterial
enzyme that can be used as a reporter. These fusions are only active when the
LacZ moiety is present in the cytoplasm (Froshauer et al., 1988).
GFP is a thoroughly tested cytoplasmic reporter that can be fused to the Cterminal end of the protein of interest (Drew et al., 2002; Rapp et al., 2004). GFP
forms a chromophore when correctly folded but can only fold in a proper manner
when on the cytoplasmic side of the inner membrane in Escherichia coli (Feilmeier
et al., 2000). Thus, if GFP fluorescence emission can be detected, a cytoplasmic
location of the reporter is indicated. When targeted to the periplasm by a Sec-type
signal peptide (Drew et al., 2002), GFP does not fold properly and does not
fluoresce (Drew et al., 2001; Feilmeier et al., 2000).
Mapping of loop regions
Mapping of loop regions in a membrane protein can be achieved by introducing
consensus sequences for protein kinase A (PKA) (Goder et al., 2000). This
cytosolic enzyme only phosphorylates extramembraneous protein domains
localized in the cytoplasm, which contain the consensus sequences. By
introducing N-glycosylation sites in membrane proteins, useful topological
information can be obtained. This will be described in more detail under
Comments of methodology.
19
Computerized topology determination
An alternative way to gain an idea of a protein’s topology is to use computer
programs that predict the topology by analyzing the amino acid sequence of the
membrane protein (Claros and von Heijne, 1994; Tusnady and Simon, 1998).
All α-helical membrane proteins have some general features like (i) the presence
of 20-30 amino acids long TM α-helices with a high overall hydrophobicity and
(ii) “the positive-inside rule”(von Heijne, 1986; von Heijne, 1992). These features
are taken into consideration when designing the prediction algorithm and
common to almost all prediction methods is that they incorporate both
hydrophobicity analysis and the positive-inside rule (von Heijne, 1992). The
topology of α-helical membrane proteins can be predicted with relatively high
accuracy from the amino acid sequence alone. It is more difficult to predict the
topology of β-barrel membrane proteins since the membrane spanning β-strands
are short and thus hard to detect in the sequence (von Heijne, 1994; von Heijne,
1996).
There are several different algorithms available for membrane protein topology
predictions. Five of the most widely used topology prediction methods are:
TMHMM (Krogh et al., 2001; Sonnhammer et al., 1998), HMMTOP (Tusnady and
Simon, 1998; Tusnady and Simon, 2001), MEMSAT (Jones et al., 1994), TOPPRED
(Claros and von Heijne, 1994; von Heijne, 1992) and PHD (Rost et al., 1996). All of
these methods are designed to identify potential TM α-helices and to predict the
overall in-out topology of the extramembraneous parts of the membrane protein.
TMHMM and HMMTOP both use a Hidden Markov model formalism to
describe the architecture of an integral membrane protein. These approaches
generally result in 65-70% correct predictions and are the best-performing
methods to date (Ikeda et al., 2001; Möller et al., 2001). MEMSAT uses dynamic
programming to optimally thread a polypeptide chain through a set of topology
models. PHD is based on a neural network predictor. TOPPRED identifies
“certain” and “putative” TM α-helices from a standard hydrophobicity plot and
then the most likely topology is suggested based on the positive inside rule (von
Heijne, 1986). There are numerous hydrophobicity scales available which can be
used for topology prediction (Chen et al., 2002; Cornette et al., 1987).
None of the topology prediction methods are perfect. For example, when a TM
helix in the analyzed protein contains charged residues, these domains are
sometimes identified as soluble loop regions. Another potential inaccuracy is
when two closely spaced TM helices, connected by a short loop, sometimes are
predicted to be one TM helix. By incorporating experimental topology
information into the prediction algorithms, the predictions are significantly
improved (Melén et al., 2003). A combination of several different topology
prediction algorithms can be used in parallel and when several of those agree on
the prediction, highly reliable topology models can be produced. Experimental
20 Marika Cassel
efforts can be focused on the less reliably predicted parts of the membrane
protein to further substantiate the topology model. Recently it has been shown
that topology prediction can be improved considerably, by constraining the
topology prediction algorithm with an experimentally determined reference point
(Daley et al., 2005). New topology prediction methods are produced at a steady
rate. However, the overall performance of these methods appears to improve
only slowly (Chen et al., 2002; Chen and Rost, 2002).
In addition to topology prediction, there are two other aspects concerning
membrane proteins that prediction methods can be applied on: (i) distinguishing
membrane proteins from soluble proteins, (ii) modelling of 3D membrane
proteins structure.
Topogenic determinants
Membrane proteins can adopt a variety of TM orientations and several factors are
involved in determining the topology. Some of these are quite well characterized,
but still many details of how topology is controlled remain to be elucidated.
Topogenic signals
It is well known that integral membrane proteins contain special sequences, socalled signal sequences or that target them to the ER membrane, where they are
cotranslationally integrated (Walter and Johnson, 1994). In addition to their
targeting function, these signals also play a role in membrane protein topogenesis
and can contribute to the final structure and function of a membrane protein.
There are four different, generally accepted kinds of signal sequences. These
include signal peptides (SP), signal-anchor (SA) sequences, reverse signal-anchor
(rSA) sequences and stop-transfer (ST) sequences (von Heijne, 1996 ; von Heijne
and Gavel, 1988).
The separate signal sequences all consist of three distinct regions: an NH2terminal hydrophilic domain (N-domain), a central hydrophobic region (Hregion) and a COOH-terminal polar region (C-domain). The ER targeting
function of the signal sequences is primarily determined by the H-region and the
orientation in the membrane is affected by the flanking regions (Nilsson and von
Heijne, 1993). The topology that a membrane protein finally adopts is partially
dependent on whether its signal sequence is cleaved off or retained as a TM
anchor during membrane protein biosynthesis.
Single-spanning membrane proteins
Single-spanning membrane proteins containing these signal sequences, can be
classified according to their orientation in the membrane (Figure 2) (von Heijne,
1996). Type I membrane proteins are oriented in the membrane with an
exoplasmic N-terminus, while the C-terminus is in the cytosol. Type I membrane
proteins are targeted to the ER membrane by a cleavable N-terminal signal
peptide (a hydrophobic stretch of 7-15 mainly apolar residues) (Blobel, 1983; von
21
Heijne, 1990) and are anchored in the membrane by a subsequent ST-sequence.
This ST-sequence is a TM spanning domain composed of around 20 hydrophobic
residues that halt a continued translocation of the polypeptide chain. The signal
peptide is cleaved off by signal peptidase on the lumenal side of the membrane.
Membrane proteins of type II have a cytoplasmic N-terminus and an exoplasmic
C-terminus. A signal-anchor sequence composed of 18-25 mainly apolar residues
anchors the protein in the membrane and induces the translocation of the Cterminal end across the membrane. Type III membrane proteins are inserted into
the lipid bilayer with an exoplasmic N- and a cytoplasmic C-terminus. This type
of membrane proteins are anchored in the membrane by a rSA-sequence that
induces translocation of the N-terminal end across the membrane. All these three
types of membrane proteins are targeted to the ER membrane in an signal
recognition particle (SRP) dependent way and are cotranslationally inserted into
the membrane by the Sec61 translocon (High and Dobberstein, 1991). Membrane
proteins of type IV are anchored to the membrane by a C-terminal hydrophobic
sequence and have their N-terminus in the cytoplasm while the C-terminus is
exoplasmic. These proteins are posttranslationally inserted into the membrane,
using an ATP-requiring mechanism that is SRP- and Sec61-independent (Kutay et
al., 1995; Kutay et al., 1993; Whitley et al., 1996).
Type I
Type II
Type III
C
N
Type IV
N
C
exoplasmic
N
C
N cytoplasmic
N
C
Figure 2. Classification of single spanning membrane proteins (adapted from Goder et al, 2001)
Charged residues
The distribution of charged residues in the sequence flanking a given TM domain
is a major factor influencing its orientation in the lipid bilayer (High and
Dobberstein, 1992). Positively charged amino acid residues are particularly
important. Statistical studies of integral membrane proteins in bacteria have
shown that positively charged amino acids are enriched in loop regions on the
cytoplasmic side of the membrane (von Heijne, 1986; von Heijne, 1989). This is
22 Marika Cassel
known as the “positive-inside rule” i.e., parts of the protein rich in lysine and/or
arginine tend to remain non-translocated. Through further statistical studies and
experiments, it has been established that the number and location of positively
charged residues are crucial for membrane protein topology (Andersson et al.,
1992; Boyd and Beckwith, 1989; Gafvelin and von Heijne, 1994; Nilsson and von
Heijne, 1990; von Heijne, 1994) and that the “positive inside rule” is a general
feature among helix-bundle proteins in a wide range of organisms and organelles,
in all three kingdoms of life (Nilsson et al., 2005; Wallin and von Heijne, 1998)
It has been shown that negatively charged residues are much less potent than
positively charged ones, and only significantly affect the topology if present in
high numbers (Nilsson and von Heijne, 1990). The importance of negatively
charged residues has been underscored though, as it was observed that they play
an active role in the membrane insertion of the small Pf3 coat protein from
Pseudomonas aeruginosa (Kiefer et al., 1997).
The membrane protein topology can also be influenced by the length and
hydrophobicity of a membrane-spanning domain (Wahlberg and Spiess, 1997). It
has further been shown that the folding state of the N-terminal domain (Denzer et
al., 1995) as well as glycosylation can contribute to the final topology.
Multi-spanning membrane proteins
Multi-spanning membrane proteins can be classified in a similar way as singlespanning proteins, based on their orientation in the membrane. The first
hydrophobic segment in multi-spanning membrane proteins acts as one of the
four kinds of signal sequences mentioned earlier (SP, SA sequence, rSA sequence
or ST sequence) and targets the nascent polypeptide chain to the ER membrane
where it initiates translocation and insertion. In some cases, the orientation of the
first TM domain dictates the orientation of the subsequent TM domains, thus
defining the overall topology of the protein. However, in most cases the final
topology result from cooperation or competition between several topogenic
determinants that are distributed throughout the polypeptide sequence (Gafvelin
et al., 1997; McGovern and Beckwith, 1991; Sato et al., 1998; Sato and Mueckler,
1999).
The impact from two topologically competing or cooperating hydrophobic
sequences on the final topology, depends on the length of the connecting loop
region (or spacer sequence) between the two TM helices. (Goder et al., 1999;
Goder and Spiess, 2001). TM domains connected by a short loop cannot orient
themselves independently of each other and are thus inserted into the membrane
as one compact unit, a helical hairpin, but with increasing spacer sequence length
the cooperativity or competition decreases (Goder and Spiess, 2001).
It has been proposed that the TM domains in a polytopic membrane protein are
kept together in the translocon and released into the membrane en bloc (Borel and
23
Simon, 1996). However, this is contradictory to cross-linking studies showing that
individual TM domains can be cross-linked to lipids as soon as they reach the
translocon, which indicates that the TM helices exit into the membrane one-byone (Heinrich et al., 2000; Martoglio et al., 1995; Mothes et al., 1997; Sadlish et al.,
2005).
Biosynthesis and targeting to the ER membrane
In eukaryotic cells most of the membrane proteins are synthesized by ER-bound
ribosomes and are then inserted into and folded in the ER membrane, from which
they are subsequently transported to the plasma membrane or other organelles
within the secretory pathway.
There are two different pathways, by which membrane proteins are targeted to
and inserted into the ER membrane. These include, (i) the co-translational
pathway which is predominant in mammalian cells and (ii) the posttranslational
pathway (Johnson and van Waes, 1999; Lecomte et al., 2003; Meacock et al., 2000;
Walter and Johnson, 1994). As mentioned earlier, the N-terminal part of newly
synthesized membrane proteins destined for the ER contains an ER targeting
signal. This signal ensures that the protein is directed to the ER membrane. In the
cotranslational pathway, the signal sequence is recognized by and bound to a
hydrophobic methionine-rich domain of the cytosolic SRP as soon as it emerges
from the ribosome (Keenan et al., 1998; Zopf et al., 1990). This event causes a
pause in protein synthesis and translation is halted until the nascent chain–
ribosome–SRP complex binds to the SRP-receptor on the rough ER (Keenan et al.,
2001). Both the SRP and the SRP receptor are GTPases that regulate the targeting
events by binding to and mediate the hydrolysis of GTP (Keenan et al., 2001). The
targeting complex then disassembles and the SRP and the SRP receptor are
released and become available for further targeting cycles (Pool et al., 2002).
Following this, the ribosome docks onto the translocon and the SP is transferred
to the recognition site in the Sec61α subunit of the translocon (Mothes et al., 1998;
Mothes et al., 1994). Here it is oriented due to topogenic determinants and
integrated into the ER membrane (Do et al., 1996; McCormick et al., 2003; Mothes
et al., 1998) (Schatz and Dobberstein, 1996). The structure of the ribosometranslocon complex shows that the exit site of the ribosomal tunnel is aligned
with the aqueous pore of the translocon (Beckmann et al., 2001). The ribosome
and lumenal chaperones provide the driving force for translocation as the
translocon channel itself is a passive conduit for polypeptides (Matlack et al.,
1998).
Several reports indicate that nascent polypeptide chains fold to various extent
while in the ribosomal tunnel (Hardesty and Kramer, 2001) and that TM domains
may be recognized already inside the ribosome. FRET analysis in combination
with cross-linking of a nascent membrane protein support this idea, indicating
24 Marika Cassel
that folding of TMs is induced in the ribosome (Woolhead et al., 2004). It has been
shown that the nascent chains interact with the ribosomal interior and thus
induce conformational changes in the highly dynamic ribosome (Frank and
Agrawal, 2000; Gao et al., 2003). These conformational changes may be
transferred to the translocon, which in turn influence the topogenesis of the
membrane protein (Tenson and Ehrenberg, 2002). This notion is supported by a
fluorescence quenching study, which shows that a TM inside the ribosome
induces conformational changes in the translocon (Liao et al., 1997).
The protein-conducting translocon
The translocon is a membrane-embedded protein-conducting channel, found in
the ER membrane of eukaryotes and in the plasma membrane of prokaryotes.
Translocons are conserved and built from the same basic components in both the
inner membrane of Escherichia coli and in the ER membrane (Rapoport et al.,
1996). The translocon works in concert with the ribosome and is responsible both
for the integration of membrane proteins and for the translocation of secretory
proteins (Figure 3) (Laird and High, 1997; Johnson and van Waes, 1999;Rapoport
et al., 2004 ;Matlack et al., 1998; von Heijne, 1997).
Figure 3. Schematic representation of a ribosome-translocon complex. Soluble proteins pass through
the translocon channel whereas TM segments of membrane proteins exit laterally into the membrane.
The structural core of the Sec61 translocon in the eukaryotic ER membrane, which
is homologous to the prokaryotic SecY translocon, is built from three different
integral membrane proteins. These include Sec61α, Sec61β and Sec61γ, where
Sec61α is the largest subunit and is thought to form the central aqueous pore
through which the nascent polypeptide chain is translocated (Crowley et al., 1994;
Gorlich and Rapoport, 1993; Johnson and van Waes, 1999; Rapoport et al., 1996;
Van den Berg et al., 2004). Sec61α is also one of the first translocon components to
interact with the polypeptide chain as it emerges from the ribosome (High et al.,
25
1993; McCormick et al., 2003; Mothes et al., 1994; Sadlish et al., 2005; Thrift et al.,
1991).
The Sec 61 complex functions in association with a variety of other membrane
proteins with specialized functions, such as oligosaccharyl transferase (OST), the
signal peptidase complex (SPase), the translocon-associated protein complex
(TRAP) and the translocating chain associated membrane protein (TRAM). OST
has its active site on the luminal side of the ER membrane where it transfers
oligosaccharides to acceptor sites for N-linked glycosylation (Asn-X-Thr) on
growing nascent polypeptide chains. The SPase removes signal peptides from
secretory proteins (Dalbey et al., 1997). TRAP is a tetrameric membrane protein
complex of unknown function (Fons et al., 2003) and TRAM is thought to function
as a chaperone during membrane protein integration as well as during early
stages of secretory protein synthesis.
The whole assembly has an outer diameter of about 100Å and the translocation
pore has a diameter of about 20Å, as shown by cryo-EM image reconstructions
(Beckmann et al., 1997; Menetret et al., 2000; Meyer et al., 1999). However, earlier
studies suggested it to be wider (Hamman et al., 1997). Electrophysiology and
fluorescence lifetime measurements have shown that the translocation pore has a
hydrophilic interior (Crowley et al., 1994; Simon and Blobel, 1991).
Whether the translocon channel forms at the interface between multiple copies of
the heterotrimeric complex or is intrinsic to a monomeric Sec61 complex, as
suggested by the recently solved X-ray structure of the archaeal SecY translocon
from Methanococcus jannaschii (Van den Berg et al., 2004), is a controversial issue.
It has been proposed that the channel is formed at the interface between three to
four copies of the Sec61/SecY complex, based on EM of the yeast and mammalian
translocons (Beckmann et al., 1997; Beckmann et al., 2001; Hamman et al., 1997;
Hanein et al., 1996; Manting et al., 2000; Menetret et al., 2000). Model building
from cryo-EM images suggests that the translocon assembly consists of four
copies of heterotrimeric Sec61 and two copies of TRAP. This study also provides
evidence for passage of the elongating chain through a single SecY heterotrimer
(Menetret et al., 2005). This is also seen in the X-ray structure of SecY (Van den
Berg et al., 2004), which suggests that a translocation pore is formed by a single
monomer of the SecY complex, rather than at the interface of multiple SecY
molecules. Recent crosslinking studies also support this model (Cannon et al.,
2005).
The structure shows that the translocon is organized in two halves where the first
half, containing TM helices 1-5, is antiparallell to the second one, containing TM
helices 6-10. The lateral gate of the translocon where hydrophobic sequences can
exit into the lipid bilayer, is found between helices 2/3 and 7/8. In the absence of
a translocating substrate, the central pore of this complex is surprisingly small
with a diameter of 3Å and is plugged by a short helix (Van den Berg et al., 2004).
26 Marika Cassel
When actively translocating, the plug would move away and the structure would
alter so as to allow the vectorial transfer of polypeptide chains. In the structure, a
ring of hydrophobic Ile residues is seen, which is thought to bind to hydrophobic
segments in the polypeptide. This ring is also thought to provide a flexible seal
around the translocating polypeptide preventing ion leakage through the
membrane (Dobberstein and Sinning, 2004; Van den Berg et al., 2004). A recent
study supports earlier indications that the ER membrane is nevertheless rather
leaky to small molecules and suggests that this may be of functional significance
for protein folding in the lumen of the ER (Le Gall et al., 2004).
Translocation
While the translocon provides a lateral exit to the lipid bilayer and rapidly orients
and integrates the TM domains emerging from the ribosome one after the other, it
simultaneously orients hydrophilic luminal and cytosolic domains of the
membrane protein. At the same time it maintains the permeability barrier of the
ER membrane, preventing ions and small molecules from freely crossing the
membrane (Gilbert et al., 2004). To date little is known regarding how the
translocon syncronizes this process. It is nevertheless clear that as the elongating
polypeptide chain passes through the channel, the translocon interprets the code
within the amino acid sequence of the nascent chain and decides whether protein
segments should be translocated through the membrane or be selected for
insertion. The basic features of this “sorting code” have recently been determined
showing that membrane insertion depends strongly on the position of polar
residues within TM segments. This study also implies that direct protein-lipid
interactions are critical during translocon-mediated membrane insertion (Hessa et
al., 2005).
Translocon-mediated membrane insertion
In several studies the environment of the polypeptide as it is integrated into the
ER membrane has been analysed in vitro showing that every TM in a polypeptide
chain is associated with the Sec61 complex during their transition into the lipid
bilayer (Heinrich and Rapoport, 2003; Laird and High, 1997; Meacock et al., 2002;
Mothes et al., 1997). The TRAM protein is also contacted by some TM domains in
polytopic membrane proteins (Meacock et al., 2002).
The integration process for polytopic membrane proteins can be described by two
theoretical models: (i) the “en masse” model and (ii) the “sequential” model
(High and Laird, 1997; Lecomte et al., 2003). In the “en masse” model, all the TM
domains of the nascent polypeptide chain are proposed to accumulate within the
ER translocon, before they are released together into the lipid bilayer. It has been
shown in one study that a polytopic protein containing 5 TM helices behaves as
though all the TM helices were in an aqueous pore (Borel and Simon, 1996).
However, this model is contradictory to the X-ray structure of SecY which
indicates that the pore may be too small to accommodate substantial numbers of
TM domains inside it (Van den Berg et al., 2004).
27
Other studies support the “sequential” model (High and Laird, 1997; Lecomte et
al., 2003), where each of the TM domains exit the Sec61 translocon individually
during the biosynthesis of the membrane protein. In a recent study, where the
integration process for the aquaporin-4 water channel (6TMs) was examined by
photo-cross-linking, it was shown that the individual TM helices in this polytopic
membrane protein are delivered from the Sec61 translocon into the lipid bilayer
in succession, where TM2 follows TM1 and so on (Sadlish et al., 2005). A recent
study of a membrane protein containing seven-TM helices has shown that the
first and second TM domains leave the translocon before the synthesis of the
protein is completed (Meacock et al., 2002). Newly synthezised TM domains may
not exit the translocon in the order of their synthesis. Yet another possibility
concerning integration of polytopic membrane proteins is that a single
polypeptide chain may have different TM domains accommodated by distinct
copies of the Sec61 complex during integration into the ER membrane
(Dobberstein and Sinning, 2004). In some cases the integration of downstream TM
domains in polytopic membrane proteins can be SRP-dependent, when for
example a long cytosolic domain preceeds a TM helix (Kuroiwa et al., 1996).
However, this is not the case for all polytopic membrane proteins (Wessels and
Spiess, 1988). It can be concluded that too few examples of the synthesis and
membrane integration processes of polytopic membrane proteins have been
studied so far to allow general principles to be formulated.
Folding of extramembraneous domains
It is still not known precisely where the soluble loops flanking TM domains are
located during synthesis, nor how their presence affects the gating of the pore. It
has been suggested that cytosolic loops could protrude out of gaps at the
cytosolic junction between the ribosome and the Sec61 complex (Beckmann et al.,
2001). A similar scenario may occur on the luminal side of the ER membrane. The
intracellular and extracellular hydrophilic regions of polytopic membrane
proteins often have important biological functions and it is important that they
are correctly folded and assembled. This is achieved by interactions with
chaperones in the ER lumen and in the cytosol that assist the folding. Examples of
lumenal chaperones are BiP and calnexin, which have been shown to be
transiently associated with polytopic membrane proteins after their synthesis
(Kopito, 1999; Swanton et al., 2003). Cytosolic chaperones Hsc70 and Hsp90 are
involved in the folding of CFTR (Chapple and Cheetham, 2003; Kopito, 1999; Loo
et al., 1998; Meacham et al., 1999; Swanton et al., 2003). It has been suggested that
specific chaperones may act to facilitate the integration of TM domains as they
exit the ER translocon (Meacock et al., 2002) and assist the folding and assembly
of the membrane spanning part of membrane proteins. However, to date only
calnexin has been implicated in this process. It associates with a misfolded
28 Marika Cassel
domain of a polytopic membrane protein preventing its degradation (Swanton et
al., 2003).
Helix-helix packing in the membrane
After being integrated into the membrane the TM helices of a polytopic
membrane protein associate with each other. Several factors are thought to be
responsible for the association of helices in membrane proteins. These include
surface complementarity, the presence of polar residues in the TM region (Choma
et al., 2000; Johnson et al., 2004; Zhou et al., 2001) and certain specific motifs such
as the well-known GXXXG pattern (MacKenzie et al., 1997; Senes et al., 2001). It is
likely that several of these factors act in concert to determine the final folded
structure, as well as the association of monomers to form an oligomer. In addition
to helix-helix interactions, interactions between the polypeptide chain with itself,
water, the bilayer and in some cases cofactors also play a role in the organization
and assembly of TM helices.
29
Comments on Methodology
Model system used for the study of helical hairpin formation
Model protein –Leader peptidase
For the study of helical hairpin formation in the ER membrane, we have used the
well-characterized protein leader peptidase (Lep) as a model membrane protein.
Lep is a 323 amino acid protease found in the inner membrane of Escherichia coli,
where it serves to remove signal peptides from secretory proteins. It contains two
TM helices (H1 and H2), a small polar domain (P1) and a large C-terminal
domain (P2) (Figure 4). The X-ray crystal structure of a catalytically active soluble
fragment of Lep has been determined at 1.9Å resolution (Paetzel et al., 1998).
Y
When expressed in vitro in the presence of dog pancreas microsomes, Lep has
been shown to insert into the microsomal membrane with the P1 loop facing the
cytoplasm and the N-terminus as well as the P2 domain in the extra-cytoplasmic
space, i.e. it adopts the same topology as in the Escherichia coli inner membrane
(Wolfe et al., 1983). The H1 segment is needed for the correct membrane insertion
and H2 is an internal uncleaved signal sequence, which is needed for
translocation of the C-terminal domain. The P2 domain contains the catalytic site.
To study helical hairpin formation, a unique acceptor site for N-linked
glycosylation (Asn-Ser-Thr) has been introduced in the P2 domain, 20 residues
downstream of H2 (Nilsson and von Heijne, 1993).
P2
X
N
H1
N
lumen
H1
Y
cy tosol
P2
Figure 4. Model protein used to study helical hairpin formation. The H2 domain of Lep has been
replaced by an artificial poly-Leu segment. Depending on the lumenal (no helical hairpin formed) or
cytoplasmic (helical hairpin formed) localization of the P2 domain, the glycosylation acceptor site will
either be modified (Y) or not ( ).
30 Marika Cassel
Manipulation of the model protein
In order to simplify our analysis of helical hairpin formation, the H2 domain of
Lep was replaced by a specifically designed poly-Leu (39L+V) TM segments, that
is long enough to form either a single TM helix or a helical hairpin (Monné et al.,
1999b). Site-directed mutagenesis was then used to introduce mutations in the
model protein.
All the mutants were expressed in vitro in presence of dog pancreas microsomes
and the proteins were analyzed by SDS-PAGE. The gels were scanned and
quantified on a phosphoimager (Fuji BAS 1000). Following this, the glycosylation
efficiency of all mutants were calculated.
For the studies in paper II and III, we introduced an extra TM segment (H3) into
the P2 domain of Lep. This H3 segment is composed of 40 Leu´s. Two
glycosylation acceptor sites were introduced in position 96 and 258. If H3 spans
the membrane once, only one acceptor site will be modified, whereas if H3 forms
a helical hairpin with a cytoplasmic turn, both sites will be exposed to the
lumenal side of the membrane and hence glycosylated. Singly and doubly
glycosylated proteins are easily distinguished by SDS-PAGE.
In paper III, the H2 TM domain of Lep was replaced by three different artificial
segments, one at a time, each compriing a stretch of hydrophobic amino acids,
containing 39LV W22 (39 Leu, 1 Val and Trp in position 22), 39LV and 30LV P18,
respectively (31 residues is close to the minimum length required for helical
hairpin formation).
Glycosylation mapping
Segments of eukaryotic membrane proteins that are translocated across the ER
membrane can become modified by N-linked glycosylation on Asn-X-Thr/Ser
acceptor sites (where X is any amino acid except proline.) Almost as soon as the
polypeptide chain enters the ER lumen, it is glycosylated on the target Asn. The
oligosaccharide is transferred onto the nascent chain in a reaction catalysed by the
enzyme OST. There is one copy of this enzyme associated with each translocon in
the ER membrane and it has its catalytic site located in the lumen of the ER
(Palade, 1975). The active site of OST is placed at a fixed distance of about 30-40 Å
from the lipid bilayer and is oriented roughly parallel to the membrane surface
(von Heijne, 1996). By introducing glycosylation acceptor sites in the protein of
interest, this defined position of the OST active site makes it possible to study
how proteins integrate into and translocate across the ER membrane. By in vitro
transcription and translation of Lep, in the presence of dog pancreas microsomes
(Liljeström and Garoff, 1991), lumenally oriented domains will be glycosylated
whereas cytoplasmically oriented domains will not be modified. An advantage of
this method is that the microsomal in vitro system closely mimics the conditions
of in vivo membrane protein assembly into the ER membrane. The presence of the
N-linked oligosaccharide can easily be detected, as glycosylated substrate
31
molecules have a lower migration rate than non-glycosylated molecules when
separated by SDS-PAGE.
The glycosylation mapping approach is a simple experimental system for the
analysis of turn formation in TM helices embedded in the ER membrane (Nilsson
et al., 1998). This technique has allowed us to perform detailed studies of the
factors that determine how helical hairpins are formed.
Alkaline extraction
To show that our poly-Leu based helical hairpins are properly assembled in the
ER membrane and to rule out that low levels of glycosylation are caused by a
failure in membrane integration rather than helical hairpin formation, we have
used sodium carbonate extraction on some of our constructs as previously
described (Hunt et al., 1997). This was done by expressing a truncated form of
several constructs forming helical hairpins, leaving H2 as the only potential
membrane-spanning segment. As expected, the proteins were not glycosylated
and were associated with the membrane pellet after alkaline extraction of the
microsomes.
Alkaline phosphatase as a topology reporter protein
In paper V we have used a combination of two topology reporter fusion methods,
where the protein under study is fused to a reporter protein, PhoA or GFP
(described below). PhoA is a commonly used reporter protein for identifying
extracellular loops of membrane proteins expressed in Escherichia coli. PhoA is
only enzymatically active on the periplasmic side of the inner membrane. In order
to be active, the protein has to form two disulfide bonds (Akiyama and Ito, 1993;
Derman and Beckwith, 1991). These two bonds can only be formed in the
oxidizing environment of the periplasm. When a substrate analogue is added to a
bacterial culture expressing the fusion construct, a chromogenic product is
formed only when the PhoA domain is in the periplasm. Its activity can be
quantified spectrophotometrically (Manoil, 1991; Manoil and Beckwith, 1986).
Green flourescent protein as a topology reporter protein
GFP is another example of a thoroughly tested topology reporter protein that can
be fused to the C-terminal part of the protein of interest. GFP forms a
chromophore when correctly folded but the protein can only fold in a proper
manner when on the cytoplasmic side of the inner membrane in Escherichia coli. A
correct folding is needed in order for GFP to fluoresce. When targeted to the
periplasm by a Sec-type signal peptid (Feilmeier et al., 2000), GFP does not fold
properly and remains non-fluorescent.
32 Marika Cassel
Results and Discussion
Formation of helical hairpins during membrane protein
integration into the ER membrane
The major aim of this thesis has been to better define the topological determinants
underlying the formation of helical hairpins in membranes. Helical hairpins are
basic folding units in membrane proteins during membrane protein assembly in
the ER membrane.
The helical hairpin is a structural unit in multi-spanning integral membrane
proteins. It is composed of two closely spaced hydrophobic TM α-helices
connected by a short turn (Engelman and Steitz, 1981). The helical hairpin is
formed during translocon assisted membrane protein integration and is thought
to be inserted into the membrane as one compact unit. It is becoming increasingly
clear that the helical hairpin is a very common structural element in membrane
proteins and a detailed understanding of its properties is of central importance.
Formation of helical hairpins with a lumenal turn, induced by
turn promoting residues (paper I)
The formation of tight turns in globular proteins has been studied for decades
both experimentally and by statistical analysis of known structures. As a result of
this research, reliable turn propensity scales have been established. However,
before we started this project nothing was really known about the residue
characteristics responsible for the formation of tight turns between transmembrane α-helices in integral membrane proteins.
Previous studies demonstrated that, when a single Pro residue is introduced near
the middle of a 40 residues long poly(Leu) TM helix, a turn is induced and the
polyLeu stretch is efficiently converted from a long, single TM helix into a helical
hairpin (Nilsson et al., 1998). Because of these findings, in paper I we extended
the study by measuring the ability of all the 20 naturally occurring amino acids to
induce the formation of a tight turn and consequently a helical hairpin, when
placed in the middle of a 40 amino acids long hydrophobic TM helix. We used a
glycosylation mapping approach that can distinguish between one long TM helix
and a helical hairpin (see previous section). This study revealed that the amino
acid residues have either a high or a low turn propensity and a turn propensity
scale for TM helices was derived. The amino acid residues with high turn
propensity are the charged (Arg, Lys, His, Glu and Asp) and the highly polar
residues (Asn and Gln) together with the classical helix breakers, Pro and Gly.
This means that these residues efficiently promote formation of helical hairpins
33
with a tight lumenal turn, when placed in the middle of a hydrophobic TM helix.
The hydrophobic amino acid residues (Leu, Phe, Ala, Met, Val and Ile), together
with the weakly polar amino acids (Ser, Thr, Tyr and Cys), have no or low turn
propensity and do not promote helical hairpin formation.
In summary, most of the results from paper I can be explained by hydrophobicity,
i.e. all hydrophobic residues prefer the membrane environment over the
membrane-water interface region and have low turn propensities. Conversely,
the charged and highly polar residues induce turn formation in order to avoid the
membrane interior. From our results we can conclude that turn propensities in
TM helices are markedly different from those of globular proteins, and in most
cases correlate closely with the hydrophobicity of the residue (Monné et al.,
1999a).
Following this, research in our laboratory was designed to derive a more finegrained turn-propensity scale. This was based on (i) measurements of the turninducing potential of single residues placed in the middle of a 31 residue
poly(Leu) segment and (ii) measurements of the turn-inducing potential of pairs
of residues placed in the middle of a 40 residue poly(Leu) segment (Monné et al.,
1999b). This study showed that if the hydrophobic segment is long enough and
contains a turn-inducing residue in the middle, it forms a helical hairpin. The
shortest TM segment that can form a helical hairpin is around 30 residues long
(Monné et al., 1999b).
Formation of helical hairpins with a cytoplasmic turn,
induced by turn promoting residues (paper II)
In previous studies, the turn of the helical hairpin was always placed on the
lumenal side of the ER membrane. To expand these studies, in paper II we
performed similar measurements of turn propensities, but in this study the turn
of the helical hairpin has a cytoplasmic location.
To study formation of helical hairpins with a cytoplasmic turn, we introduced
one or a pair of each of the 20 naturally occurring amino acids in the middle of
the polyLeu stretch (H3). Subsequently, their ability to induce a cytoplasmic turn
was assessed. The result showed that a single Pro residue induces a helical
hairpin in only 36% of the molecules and 3 Pro are needed to reach full degree of
helical hairpin formation. This is in contrast to our previous studies of lumenal
turns, where one Pro was sufficient to induce full helical hairpin formation.
We demonstrated that none of the singly inserted residues were able to promote
efficient formation of a helical hairpin with a cytoplasmic turn to any significant
extent. However, charged and polar amino acids all gave some degree of helical
hairpin formation. Pairs of polar or charged residues had a higher turn inducing
34 Marika Cassel
effect but the molecules do not reach full helical hairpin formation in most cases.
All the hydrophobic residues plus Thr, Ser, Cys, Gly, Trp and Tyr had low or no
turn-inducing effect even when placed in tandem. Only in constructs with 2 Lys
or 2 Arg in the middle, a full degree of helical hairpins was formed.
To summarize, from the results in paper I and paper II we can conclude that
hydrophobic amino acids do not induce turns, charged residues are strong turn
promoters and most polar amino acids are in-between. Among the non-charged
residues, Pro and Asn have the highest turn propensities.
This general picture holds true both for helical hairpins with turns on the
cytoplasmic and lumenal side of the ER membrane. However, there are some
differences. In the case of turns on the cytoplasmic side of the ER membrane, a
higher number of turn-promoting residues are required to induce a helical
hairpin. Arg and Lys are the strongest turn-promoters on the cytoplasmic side,
but on the lumenal side, Pro, Asn and Arg are the strongest ones. The high
cytoplasmic turn propensities for Arg and Lys may be related to the positive
inside rule.
The differences in the number of turn–promoting residues required to induce a
helical hairpin with a cytoplasmic respectively a luminal turn may be an effect of
the translocon mediated insertion event. During insertion into the ER membrane
an internal signal-anchor sequence like H2 presumably enters the translocon in a
loop or hairpin conformation which may facilitate the formation of a permanent
helical hairpin in the presence of a turn-promoting residue. The H3 stop-transfer
sequence on the other hand presumably enters the translocon in a stretched
conformation and the effect of turn-promoting residues may be weaker. This may
explain why a smaller number of turn-promoting residues are required when the
turn is on the lumenal side.
Helical hairpin formation promoted by flanking charged
residues (paper III)
In paper III, we shifted our attention to the possible role of the residues that flank
the TM helices. As charged residues are known to act as powerful topological
determinants, in both prokaryotic and eukaryotic membrane proteins, we
considered the possibility that charged residues in the flanking region of a long
hydrophobic TM helix may specifically affect helical hairpin formation. The
results from paper III showed that both Lys and Asp residues, introduced in the
C-terminally flanking region, promote the formation of helical hairpins with a
lumenally oriented turn. A helical hairpin can thus be induced even in a
uniformly hydrophobic segment, provided that the number of C-terminally
flanking charged residues is sufficiently high. At the same time, N-terminally
35
flanking residues have no effect on helical hairpin formation in our model
protein.
It was further shown that C-terminally flanking Lys and Asp residues inhibit the
formation of helical hairpins with a cytoplasmic turn. The effect of Lys was
somewhat stronger than that of Asp, which is in accordance with the positive
inside rule. The effect of increasing the separation between clusters of charged
residues (four Lys or four Asp) and the hydrophobic stretch for helical hairpins
with lumenal and cytoplasmic turn was also studied. We found that a cluster of
four consecutive Asp or Lys residues can affect the efficiency of helical hairpin
formation even when placed 30 residues or more downstream of the hydrophobic
stretch. This may suggest that when the helical hairpin forms in the translocon,
the whole length of the nascent chain present in the ribosomal tunnel (about 40
residues) at the time can have an impact on the process of helical hairpin
formation.
Helical hairpin formation induced by inter-helical hydrogen
bonds (paper IV)
As mentioned previously, the polypeptide chain is threaded through the
translocon during membrane protein biosynthesis. In this process, hydrophobic
regions of the nascent chain are recognised by the translocon and transferred
laterally into the membrane, either one by one, or associated in pairs (Do et al.,
1996; Heinrich et al., 2000; Kanki et al., 2003; Oliver et al., 1995) or as higher–order
multimers (Borel and Simon, 1996). Several factors are thought to be responsible
for the association of TM helices in membrane proteins, including surface
complementarity, the GXXXG-motif (MacKenzie et al., 1997; Senes et al., 2001)
and the presence of polar residues in the TM region. (Choma et al., 2000; Johnson
et al., 2004; Zhou et al., 2001). It is likely that several of these factors act in concert
to determine the final folded structure of membrane proteins. However, before
the membrane protein folding pathway is fully understood many details remain
to be elucidated of how and to what extent different interactions direct the
folding and association of TM segments.
In several studies, it has been shown that inter-helical hydrogen bond formation
between pairs of the polar residues Asn or Asp in TM α-helices can drive efficient
oligomerization in detergent micelles, model membranes, and in the Escherichia
coli inner membrane (Choma et al., 2000; Gratkowski et al., 2001; Howard et al.,
2002). These observations inspired us to examine whether pairs of Asn or Asp
residues in a model TM helix also could promote the formation of helical hairpins
during membrane protein assembly into the ER membrane.
36 Marika Cassel
For the study in paper IV, one or a pair of Asn or Asp residues were introduced
in different positions in a poly(Leu) stretch in the H2 position of Lep. Helical
hairpin formation was assayed using the glycosylation mapping approach. Our
results showed that none of the single Asn-mutations induced efficient helical
hairpin formation, while single Asp-mutations have some effect. We further
showed that interhelical hydrogen bonds between Asn-Asn and Asp-Asp pairs
can drive helical hairpin formation. Asn-Asn and Asp-Asp pairs increase the
efficiency of helical hairpin formation by 1.5-2-fold over that seen for the
corresponding single Asn or Asp residues. From our results, it is also evident that
the effects on helical hairpin formation by Asn-Asn and Asp-Asp pairs are highly
position specific.
From the results of paper IV, we can conclude that the helical hairpin
conformation is stabilized by hydrogen bonding. The results further imply that
the formation of helical hairpins must take place in a non-aqueous environment
in the Sec61 translocon, since stable inter-residue hydrogen bonding would not be
expected in an aqueous environment. Instead residues would prefer to form
hydrogen bonds with the surrounding water molecules. A non-aqueous
environment in the Sec61 translocon has been implied in a previous study
(Nilsson et al., 2003). The results further implies that the two halves of the helical
hairpin formed in the TM segment cannot rotate freely relative to one another, as
only residues in positions on one face of the two helices are implicated.
Confronting fusion protein-based membrane protein
topology mapping with reality: the Escherichia coli ClC
chloride channel (paper V)
In the most recent study of this thesis, a detailed fusion-based topology mapping
of the Escherichia coli chloride channel YadQ was performed. This was done to
evaluate GFP and PhoA topology mapping assays and topology prediction
methods.
The X-ray structure of YadQ was reported in 2002 (Dutzler et al., 2002). YadQ is a
homodimer, with an ion pore in each of the monomers (Figure 5). Each monomer
contains 14 TM helices of varying lengths of which some are highly inclined
relative to the membrane normal. Considering the complicated structural
arrangement of the α-helices, this protein was considered to be a challenge for
topology mapping by reporter fusions.
From the YadQ structure optimal fusion joints were chosen and a set of fusions
were made to each loop in the structure. The reporter fusion activities were
mapped onto the 3D structure of YadQ and on the corresponding topology map.
We found that the reporter fusion data accurately reflects the topology of 10 of
the 14 TM helices, but do not score two short, weakly hydrophobic helical
37
hairpins on the periphery of the protein. The fully constrained topology
prediction method TMHMM only predicts 4 of the 7 helical hairpins correctly. We
suggest that the reporter fusion results reflect not only the final topology of the
protein but also pin-point parts parts of the protein that may insert into the
membrane at a late stage in the folding process. The reporter fusion approach
thus offer new insight into the folding pathway of YadQ.
Figure 5. 3D structure of the YadQ homodimer. The two subunits are shaded in gold and white. The
periplasmic side is up.
38 Marika Cassel
Concluding Remarks
We have shown that the formation of helical hairpins during membrane protein
integration into the ER membrane can be dependent both on the overall length of
the hydrophobic segment and on the amino acids flanking the TM segment.
Furthermore, the identity of the central, potentially turn-forming residues is of
importance. Thus, charged, polar, and classic helix-breaking residues are good
turn-formers and effectively induce helical hairpin formation when placed near
the middle of a sufficiently long hydrophobic stretch. In contrast, centrally placed
apolar residues do not induce helical hairpin formation, but rather cause the
hydrophobic stretch to insert as a single, long TM helix that spans the membrane
once.
Hydrophobic amino acids do not induce turns, charged residues are strong turn
promoters and most polar amino acids are in-between. Among the non-charged
residues, Pro and Asn have the highest turn propensities. This general picture
holds both for helical hairpins with turns on the cytoplasmic and lumenal side of
the ER membrane. However, a higher number of turn-promoting residues are
required to induce a helical hairpin with a cytoplasmic turn. Arg and Lys are the
strongest turn-promoters on the cytoplasmic side of the ER membrane, and on the
lumenal side, Pro, Asn and Arg are the strongest ones.
C-terminally flanking Lys and Asp residues promote the formation of helical
hairpins with a lumenal turn, while N-terminally flanking residues have no effect
on helical hairpin formation. In contrast, C-terminally flanking Lys and Asp
residues inhibit the formation of helical hairpins with a cytoplasmic turn. The
effect on helical hairpin formation of C-terminally flanking Lys and Asp residues
decrease with the distance from the hydrophobic stretch. We have also shown
that interhelical hydrogen bonds between Asn-Asn and Asp-Asp pairs can drive
helical hairpin formation in a position-specific manner.
Some of the topological determinants underlying the formation of helical hairpins
during membrane protein integration into the ER membrane have been clarified
in this thesis. These observations, together with future results, will increase our
understanding of what determines membrane protein topology and structure. In
addition, the information obtained may be relevant for improving current
methods of membrane protein topology prediction.
39
Acknowledgements
Många är ni som förgyllt min tillvaro under doktorandtiden och fått mig att nå ända
fram. Ett speciellt VARMT TACK till:
Gunnar, min handledare, för all din hjälp och uppmuntan som jag fått under
doktoreringen. Tack, för att du lotsat mig i rätt riktning och för att du gett mig frihet i
arbetet, och för att du alltid blivit så glad och entusiastisk, även för de allra minsta
resultat som jag lyckats frambringa. Tack också till både dig och Anna för trevliga
middagar och annandags-fika!
Stefan, för ditt stöd och uppmuntran och för att du är så juste och bryr dig om och
gör ett så himla bra jobb som institutionschef.
Anki, Ann, Kicki, Bogos, Eddie och Peter för att ni är så vänliga och hjälpsamma och
alltid ställer upp.
Mikaela, för att du accepterat att ständigt bli ihopblandad med mig. För ditt ständiga
stöd och din positiva syn. För alla oändligt många gånger du fått mig att skratta. För
att du är en god vän och pärla i största allmänhet.
Karin, för att du är min bästa lunchkompis och en god vän, som alltid är snäll och
vänlig och ger stöd. Tack för alla mysiga o trevliga fikor under mammaledigheten
och tack för allt kul på Korsika. Tack också för hjälp med sista peket.
Malin, för att du är så snäll och bryr dig om. För att du lyssnar och stöttar. För allt
kul vi hade på Korsika. Tack till både dig och Johan för mysiga supermiddagar! Sån´t
får det bli mer av.
Annika, min vän och fd labkompis. Vilket samarbete vi hade! Tack för att du alltid
stöttat mig och bryr dig om. Tack för allt kul vi haft. Önskar att du kunde flytta
tillbaka hem snart igen!
Magnus, för att du introducerade mig i labbandet, för att du fick mig att ”förstå mitt
eget bästa” och ta klivet att doktorera. Tack för allt kul vi haft på den goda tiden då
du fortfarande var kvar i gruppen.
Tara, för ditt stöd och din omtanke och all hjälp och framförallt för allt kul vi haft.
Mina labkompisar: Marie, Joy, Dan, Carolina, Filippa, Susanna, Yoko, för all er hjälp
och för all den kunskap ni delar med er av, men framförallt tack för att ni är så
vänliga och gör dagarna på labbet extra roliga.
Louise, David, Sam och David och Jan-Willem för att ni är så trevliga labgrannar och
bjuder på så mycket goda grejor.
40 Marika Cassel
Alla ni som tidigare har varit i min grupp, för all er hjälp och kunskap ni delar med
er av: Marie, Chen-Ni, Nadja, Thomas, Susana, Susanna, Paula
IngMarie, för att du alltid är hjälpsam. Din arbetsamhet och ditt vetenskapliga
engagemang är inspirerande.
Åke, min bihandledare, för att du varit nyfiken på och intresserat dig för min
forskning.
Marlene, för att du är en så god vän som jag tycker så mycket om.
Anna, Linus och Alice, för att ni är så härliga och omtänksamma och vet hur livet ska
levas.
Camilla, Lars och Isak, Sofia, Fredrik och Eskil, Ingeborg, Magnus och Melker, för att
ni förgyller tillvaron med vänskap och goda middagar.
Framför allt vill jag tacka min underbara familj:
Mina svärföräldrar: Margareta och Lars, Claes och Marie för att ni är så varma,
generösa och fantastiska människor.
Calle, för att du är så juste, rolig, snäll och hjälpsam. Kalle o Elin, för att ni är så
snälla och gulliga och bryr er om och för att det är så kul att vara med er. Samt resten
av bonusfamiljen: Caroline, Paul, Daisy, Louisa, Thomas, Clara, Jakob, Christine,
Emmy och Emil, vilken familj!
Mamma och Pappa, för er outtömliga omtanke, värme och godhet. Tack för alla
oändligt många gånger som ni ställt upp för mig och Tobias. Tack för att ni alltid tror
på mig och ger mig stöd i mot- och medgång.
Stellan, världens bästa bror, för allt ditt stöd. För att du är så genuint snäll och bryr
dig om. Tack för att du får mig att tänka på härliga saker som segling och havet.
Filippa, söta lilla solstråle, som är så härligt glad, god och empatisk. Du skänker så
mycket glädje och lycka och får oss att skratta. Du är en pärla.
Tobias, min Älskling. Det svåraste med den här avhandlingen har faktiskt varit att
komma på hur jag i ord skall kunna tacka Dig... för all oändlig kärlek, lycka och
glädje som Du ger mig, för att Du gör livet så härligt att leva, för all hjälp och allt Ditt
stöd, för att Du får mig att skratta. Du har gett mig allt som jag någonsin drömt om.
Du är helt fantastisk, en pärla av stora mått. Du är mitt allt.
41
References
Akiyama, Y. and Ito, K. (1993) Folding and
assembly of bacterial alkaline phosphatase in
vitro and in vivo. J Biol Chem, 268, 8146-8150.
Bowie, J.U. (2000) Understanding membrane
protein structure by design. Nat Struct Biol, 7,
91-94.
Andersson, H., Bakker, E. and von Heijne, G.
(1992) Different positively charged amino
acids have similar effects on the topology of a
polytopic transmembrane protein in Escherichia
coli. J Biol Chem, 267, 1491-1495.
Boyd, D. and Beckwith, J. (1989) Positively
charged amino acid residues can act as
topogenic determinants in membrane proteins.
Proc Natl Acad Sci USA, 86, 9446-9450.
Arora, A., Abildgaard, F., Bushweller, J.H.
and Tamm, L.K. (2001) Structure of outer
membrane protein A transmembrane domain
by NMR spectroscopy. Nat Struct Biol, 8, 334338.
Beckmann, R., Bubeck, D., Grassucci, R.,
Penczek, P., Verschoor, A., Blobel, G. and
Frank, J. (1997) Alignment of conduits for the
nascent polypeptide chain in the ribsomeSec61 complex. Science, 278, 2123-2126.
Beckmann, R., Spahn, C.M., Eswar, N.,
Helmers, J., Penczek, P.A., Sali, A., Frank, J.
and Blobel, G. (2001) Architecture of the
protein-conducting channel associated with
the translating 80S ribosome. Cell, 107, 361-372.
Belrhali, H., Nollert, P., Royant, A., Menzel,
C., Rosenbusch, J. P., Landau, E. M. and
Pebay-Peyroula, E. (1999) Protein, lipid and
water organization in bacteriorhodopsin
crystals: a molecular view of the purple
membrane at 1.9 A resolution. Structure Fold
Des, 7, 909-917
Blobel, G. (1983) Control of intracellular
protein traffic. Methods Enzymol, 96, 663-682.
Borel, A.C. and Simon, S.M. (1996) Biogenesis
of polytopic membrane proteins: membrane
segments assemble with translocation channels
prior to membrane integration. Cell, 85, 379389.
Bowie, J.U. (1997) Helix packing in membrane
proteins. J Mol Biol, 272, 780-789.
Boyd, D., Traxler, B. and Beckwith, J. (1993)
Analysis of the topology of a membrane
protein by using a minimum number of
alkaline phosphatase fusions. J Bacteriol, 175,
553-556.
Broome-Smith, J.K. and Spratt, B.G. (1986) A
vector for the construction of translational
fusions to TEM ß-lactamase and the analysis of
protein export signals and membrane protein
topology. Gene, 49, 341-349.
Broome-Smith, J.K., Tadayyon, M. and
Zhang, Y. (1990) Beta-Lactamase As a Probe of
Membrane Protein Assembly and Protein
Export. Mol Microbiol, 4, 1637-1644.
Brändén, C. and Tooze, J. (1991) Introduction to
Protein Structure. Garland Publishing, New
York.
Buchanan, S.K. (1999) β-barrel proteins from
bacterial outer membranes: structure, function
and refolding. Curr Opin Struct Biol, 9, 455-461.
Cannon, K.S., Or, E., Clemons, W.M., Jr.,
Shibata, Y. and Rapoport, T.A. (2005)
Disulfide bridge formation between SecY and
a translocating polypeptide localizes the
translocation pore to the center of SecY. J Cell
Biol, 169, 219-225.
Chapple, J.P. and Cheetham, M.E. (2003) The
chaperone environment at the cytoplasmic face
of the endoplasmic reticulum can modulate
rhodopsin processing and inclusion formation.
J Biol Chem, 278, 19087-19094.
42 Marika Cassel
Chen, C.P., Kernytsky, A. and Rost, B. (2002)
Transmembrane helix predictions revisited.
Protein Sci, 11, 2774-2791.
Chen, C.P. and Rost, B. (2002) Long
membrane helices and short loops predicted
less accurately. Protein Sci, 11, 2766-2773.
Chiu, M.L., Nollert, P., Loewen, M.C.,
Belrhali, H., Pebay-Peyroula, E., Rosenbusch,
J.P. and Landau, E.M. (2000) Crystallization in
cubo: general applicability to membrane
proteins. Acta Crystal D Biol Crystal, 56 ( Pt 6),
781-784.
enzymology of the type I signal peptidases.
Protein Sci, 6, 1129-1138.
Daley, D.O., Rapp, M., Granseth, E., Melén,
K., Drew, D. and von Heijne, G. (2005) Global
topology analysis of the Escherichia coli inner
membrane proteome. Science, 308, 1321-1323.
Deisenhofer, J., Epp, O., Miki, K., Huber, R.
and Michel, H. (1985) Structure of the protein
subunits in the photosynthetic reaction centre
of Rhodopseudomonas viridis at 3Å resolution.
Nature, 318, 618-624.
Choma, C., Gratkowski, H., Lear, J.D. and
DeGrado, W.F. (2000) Asparagine-mediated
self-association of a model transmembrane
helix. Nat Struct Biol, 7, 161-166.
Denzer, A.J., Nabholz, C.E. and Spiess, M.
(1995) Transmembrane orientation of signalanchor proteins is affected by the folding state
but not the size of the N- terminal domain.
EMBO J, 14, 6311-6317.
Claros, M.G. and von Heijne, G. (1994)
TopPred II: An improved software for
membrane protein structure prediction.
CABIOS, 10, 685-686.
Derman, A.I. and Beckwith, J. (1991)
Escherichia coli alkaline phosphatase fails to
acquire disulfide bonds when retained in the
cytoplasm. J Bacteriol, 173, 7719-7722.
Cornette, J.L., Cease, K.B., Margalit, H.,
Spouge, J.L., Berzofsky, J.A. and DeLisi, C.
(1987) Hydrophobicity scales and
computational techniques for detecting
amphipathic structures in proteins. J Mol Biol,
195, 659-685.
Do, H., Falcone, D., Lin, J., Andrews, D.W.
and Johnson, A.E. (1996) The cotransational
integration of membrane proteins into the
phospholipid bilayer is a multistep process.
Cell, 85, 369-378.
Cowan, S.W. and Rosenbusch, J.P. (1994)
Folding pattern diversity of integral membrane
proteins. Science, 264, 914-916.
Cowan, S.W., Schirmer, T., Rummel, G.,
Steiert, M., Ghosh, R., Pauptit, R.A.,
Joansonius, J.N. and Rosenbusch, J.P. (1992)
Crystal structures explain functional
properties of two E. coli porins. Nature, 358,
727-733.
Crowley, K.S., Liao, S., Worrell, V.E.,
Reinhart, G.D. and Johnson, A.E. (1994)
Secretory proteins move through the
endoplasmic reticulum membrane via an
aqueous, gated pore. Cell, 78, 461-471.
Dalbey, R.E., Lively, M.O., Bron, S. and Van
Dijl, J.M. (1997) The chemistry and
Dobberstein, B. and Sinning, I. (2004)
Structural biology. Surprising news from the
PCC. Science, 303, 320-322.
Drew, D., Sjöstrand, D., Nilsson, J., Urbig, T.,
Chin, C.N., de Gier, J.W. and von Heijne, G.
(2002) Rapid topology mapping of Escherichia
coli inner-membrane proteins by prediction
and PhoA/GFP fusion analysis. Proc Natl Acad
Sci USA, 99, 2690-2695.
Drew, D., von Heijne, G., Nordlund, P. and
de Gier, J.W.L. (2001) Green fluorescent
protein as an indicator to monitor membrane
protein overexpression in Escherichia coli. FEBS
Letters, 507, 220-224.
Dutzler, R., Campbell, E.B., Cadene, M.,
Chait, B.T. and MacKinnon, R. (2002) X-ray
structure of a ClC chloride channel at 3.0 Å
43
reveals the molecular basis of anion selectivity.
Nature, 415, 287-294.
Engelman, D.M. and Steitz, T.A. (1981) The
spontaneous insertion of proteins into and
across membranes: The helical hairpin
hypothesis. Cell, 23, 411-422.
Feilmeier, B.J., Iseminger, G., Schroeder, D.,
Webber, H. and Phillips, G.J. (2000) Green
fluorescent protein functions as a reporter for
protein localization in Escherichia coli. J
Bacteriol, 182, 4068-4076.
Fernandez, C., Hilty, C., Bonjour, S.,
Adeishvili, K., Pervushin, K. and Wuthrich,
K. (2001) Solution NMR studies of the integral
membrane proteins OmpX and OmpA from
Escherichia coli. FEBS Lett, 504, 173-178.
Fernandez, C., Hilty, C., Wider, G. and
Wuthrich, K. (2002) Lipid-protein interactions
in DHPC micelles containing the integral
membrane protein OmpX investigated by
NMR spectroscopy. Proc Natl Acad Sci U S A,
99, 13533-13537.
Fons, R.D., Bogert, B.A. and Hegde, R.S.
(2003) Substrate-specific function of the
translocon-associated protein complex during
translocation across the ER membrane. J Cell
Biol, 160, 529-539.
Frank, J. and Agrawal, R.K. (2000) A ratchetlike inter-subunit reorganization of the
ribosome during translocation. Nature, 406,
318-322.
Froshauer, S., Green, G.N., Boyd, D.,
McGovern, K. and Beckwith, J. (1988) Genetic
analysis of the membrane insertion and
topology of MalF, a cytoplasmic membrane
protein of Escherichia coli. J Mol Biol, 200, 501511.
Gafvelin, G., Sakaguchi, M., Andersson, H.
and von Heijne, G. (1997) Topological rules
for membrane protein assembly in eukaryotic
cells. J Biol Chem, 272, 6119-6127.
Gafvelin, G. and von Heijne, G. (1994)
Topological "frustration" in multi-spanning E.
coli inner membrane proteins. Cell, 77, 401-412.
Gao, H., Sengupta, J., Valle, M., Korostelev,
A., Eswar, N., Stagg, S.M., Van Roey, P.,
Agrawal, R.K., Harvey, S.C., Sali, A.,
Chapman, M.S. and Frank, J. (2003) Study of
the structural dynamics of the E coli 70S
ribosome using real-space refinement. Cell,
113, 789-801.
Gilbert, R.J., Fucini, P., Connell, S., Fuller,
S.D., Nierhaus, K.H., Robinson, C.V.,
Dobson, C.M. and Stuart, D.I. (2004) Threedimensional structures of translating
ribosomes by Cryo-EM. Mol Cell, 14, 57-66.
Goder, V., Bieri, C. and Spiess, M. (1999)
Glycosylation can influence topogenesis of
membrane proteins and reveals dynamic
reorientation of nascent polypeptides within
the translocon. J Cell Biol, 147, 257-266.
Goder, V., Crottet, P. and Spiess, M. (2000) In
vivo kinetics of protein targeting to the
endoplasmic reticulum determined by sitespecific phosphorylation. EMBO J, 19, 67046712.
Goder, V. and Spiess, M. (2001) Topogenesis
of membrane proteins: determinants and
dynamics. FEBS Lett, 504, 87-93.
Gorlich, D. and Rapoport, T.A. (1993) Protein
translocation into proteoliposomes
reconstituted from purified components of the
endoplasmic reticulum membrane. Cell, 75,
615-630.
Gouaux, E. (1997) Channel-forming toxins:
tales of transformation. Curr Opin Struct Biol, 7,
566-573.
Gratkowski, H., Lear, J.D. and DeGrado, W.F.
(2001) Polar side chains drive the association of
model transmembrane peptides. Proc Natl Acad
Sci U S A, 98, 880-885.
Hamman, B., Chen, J.-C., Johnson, E. and
Johnson, A. (1997) The aqueous pore through
44 Marika Cassel
the translocon has a diameter of 40-60 Å
during cotrsnaltional protein translocation at
the ER membrane. Cell, 89, 535-544.
Hanein, D., Matlack, K.E.S., Jungnickel, B.,
Plath, K., Kalies, K.-U., Miller, K.R.,
Rapoport, T. and Akey, C.W. (1996)
Oligomeric rings of the Sec61p complex
induced by ligands required for protein
translocation. Cell, 87, 721-732.
Hardesty, B. and Kramer, G. (2001) Folding of
a nascent peptide on the ribosome. Prog Nucleic
Acid Res Mol Biol, 66, 41-66.
Heinrich, S., Mothes, W., Brunner, J. and
Rapoport, T. (2000) The Sec61p complex
mediates the integration of a membrane
protein by allowing lipid partitioning of the
transmembrane domain. Cell, 102, 233-244.
Heinrich, S.U. and Rapoport, T.A. (2003)
Cooperation of transmembrane segments
during the integration of a double-spanning
protein into the ER membrane. EMBO J, 22,
3654-3663.
Henderson, R., Baldwin, J.M., Ceska, T.A.,
Zemlin, F., Beckmann, E. and Downing, K.H.
(1990) A Model for the structure of
bacteriorhodopsin based on high resolution
electron cryo-microscopy. J Mol Biol, 213, 899929.
Henderson, R. and Unwin, P.N.T. (1975)
Three-dimensional model of purple membrane
obtained by electron microscopy. Nature, 257,
28-32.
Hessa, T., Kim, H., Bihlmaier, K., Lundin, C.,
Boekel, J., Andersson, H., Nilsson, I., White,
S.H. and von Heijne, G. (2005) Recognition of
transmembrane helices by the endoplasmic
reticulum translocon. Nature, 433, 377-381.
High, S. and Dobberstein, B. (1991) The Signal
Sequence Interacts with the Methionine-Rich
Domain of the 54-kD Protein of Signal
Recognition Particle. J Cell Biol, 113, 229-233.
High, S. and Dobberstein, B. (1992)
Mechanisms that determine the
transmembrane disposition of proteins. Curr
Opin Cell Biol, 4, 581-586.
High, S. and Laird, V. (1997) Membrane
protein biosynthesis - all sewn up? Trends Cell
Biol., 7, 206-210.
High, S., Martoglio, B., Görlich, D.,
Andersen, S.S.L., Ashford, A.J., Giner, A.,
Hartmann, E., Prehn, S., Rapoport, T.A.,
Dobberstein, B. and Brunner, J. (1993) Sitespecific photocrosslinking reveals that Sec61p
and TRAM contact different regions of a
membrane-inserted signal sequence. J Biol
Chem, 268, 26745-26751.
Howard, K.P., Lear, J.D. and DeGrado, W.F.
(2002) Sequence determinants of the energetics
of folding of a transmembrane four-helixbundle protein. Proc Natl Acad Sci U S A, 99,
8568-8572.
Hunt, J., Rath, P., Rotschild, K. and
Engelman, D. (1993) Spontaneous, pHdependent membrane insertion of a
transbilayer α-helix. Biochemistry, 36, 1517715192.
Hunt, J.F., Rath, P., Rothschild, K.J. and
Engelman, D.M. (1997) Spontaneous, pHdependent membrane insertion of a
transbilayer α-helix. Biochemistry, 36, 1517715192.
Hwang, P.M., Choy, W.Y., Lo, E.I., Chen, L.,
Forman-Kay, J.D., Raetz, C.R., Prive, G.G.,
Bishop, R.E. and Kay, L.E. (2002) Solution
structure and dynamics of the outer membrane
enzyme PagP by NMR. Proc Natl Acad Sci U S
A, 99, 13560-13565.
Ikeda, M., Arai, M., Lao, D. and Shimizu, T.
(2001) Transmembrane topology prediction
methods: A re-assessment and improvement
by a consensus method using a data-set of
experimentally characterized transmembrane
topologies. In Silico Biol, 2, 1-15.
45
Ikeda, M., Arai, M., Okuno, T. and Shimizu,
T. (2003) TMPDB: a database of
experimentally-characterized transmembrane
topologies. Nucl Acids Res, 31, 406-409.
Johnson, A.E. and van Waes, M.A. (1999) The
translocon: A dynamic gateway at the ER
membrane. Annu Rev Cell Dev Biol, 15, 799-842.
Johnson, R.M., Heslop, C.L. and Deber, C.M.
(2004) Hydrophobic helical hairpins: design
and packing interactions in membrane
environments. Biochemistry, 43, 14361-14369.
Jones, D.T., Taylor, W.R. and Thornton, J.M.
(1994) A model recognition approach to the
prediction of all-helical membrane protein
structure and topology. Biochemistry, 33, 30383049.
Kaback, H.R., Voss, J. and Wu, J.H. (1997)
Helix packing in polytopic membrane
proteins: The lactose permease of Escherichia
coli. Curr Opin Struct Biol, 7, 537-542.
Kanki, T., Young, M.T., Sakaguchi, M.,
Hamasaki, N. and Tanner, M.J.A. (2003) The
N-terminal region of the transmembrane
domain of human erythrocyte band 3 Residues critical for membrane insertion and
transport activity. J Biol Chem, 278, 5564-5573.
Keenan, R.J., Freymann, D.M., Stroud, R.M.
and Walter, P. (2001) The signal recognition
particle. Annual Review of Biochemistry, 70, 755775.
Keenan, R.J., Freymann, D.M., Walter, P. and
Stroud, R.M. (1998) Crystal structure of the
signal sequence binding subunit of the signal
recognition particle. Cell, 94, 181-191.
Krantz, B.A., Melnyk, R.A., Zhang, S., Juris,
S.J., Lacy, D.B., Wu, Z., Finkelstein, A. and
Collier, R.J. (2005) A phenylalanine clamp
catalyzes protein translocation through the
anthrax toxin pore. Science, 309, 777-781.
Krogh, A., Larsson, B., von Heijne, G. and
Sonnhammer, E. (2001) Predicting
transmembrane protein topology with a
hidden Markov model. Application to
complete genomes. J Mol Biol, 305, 567-580.
Kuroiwa, T., Sakaguchi, M., Omura, T. and
Mihara, K. (1996) Reinitiation of protein
translocation across the endoplasmic reticulum
membrane for the topogenesis of
multispanning membrane proteins. J Biol
Chem, 271, 6423-6428.
Kutay, U., Ahnert-Hilger, G., Hartmann, E.,
Wiedenmann, B. and Rapoport, T. (1995)
Transport route for synaptobrevin via a novel
pathway for insertion into the endoplasmic
reticulum. EMBO J, 14, 217-223.
Kutay, U., Hartmann, E. and Rapoport, T.A.
(1993) A class of membrane proteins with a Cterminal anchor. Trends Cell Biol, 3, 72-75.
Laird, V. and High, S. (1997) Discrete crosslinking products identified during membrane
protein biosynthesis. J Biol Chem, 272, 19831989.
Landau, E.M. and Rosenbusch, J.P. (1996)
Lipidic cubic phases: a novel concept for the
crystallization of membrane proteins. Proc Natl
Acad Sci U S A, 93, 14532-14535.
Lanyi, J.K. (2004) X-ray diffraction of
bacteriorhodopsin photocycle intermediates.
Mol Membr Biol, 21, 143-150.
Kiefer, D., Hu, X.T., Dalbey, R. and Kuhn, A.
(1997) Negatively charged amino acid residues
play an active role in orienting the Secindependent Pf3 coat protein in the Escherichia
coli inner membrane. EMBO J., 16, 2197-2204.
Laude, A.J. and Prior, I.A. (2004) Plasma
membrane microdomains: organization,
function and trafficking. Mol Membr Biol, 21,
193-205.
Kopito, R.R. (1999) Biosynthesis and
degradation of CFTR. Physiol Rev, 79, S167-173.
Le Gall, S., Neuhof, A. and Rapoport, T.
(2004) The endoplasmic reticulum membrane
46 Marika Cassel
is permeable to small molecules. Mol Biol Cell,
15, 447-455.
Lecomte, F.J., Ismail, N. and High, S. (2003)
Making membrane proteins at the mammalian
endoplasmic reticulum. Biochem Soc Trans, 31,
1248-1252.
Levy, D. (1996) Membrane proteins which
exhibit multiple topological orientations.
Essays Biochem, 31, 49-60.
Liao, S., Lin, J., Do, H. and Johnson, A. (1997)
Both lumenal and cytosolic gating of the
aqueous ER translocon pore are regulated
from inside the ribosome during membrane
protein integration. Cell, 90, 31-41.
Liljeström, P. and Garoff, H. (1991) Internally
located cleavable signal sequences direct the
formation of Semliki Forest virus membrane
proteins from a polyprotein precursor. J Virol,
65, 147-154.
Manoil, C., Mekalanos, J.J. and Beckwith, J.
(1990) Alkaline Phosphatase Fusions - Sensors
of Subcellular Location. J Bact, 172, 515-518.
Manting, E.H., van der Does, C., Remiy, H.,
Engel, A. and Driessen, A.J.M. (2000) SecYEG
assembles into a tetramer to form the active
protein translocation channel. EMBO J, 19, 852861.
Martoglio, B., Hofmann, M.W., Brunner, J.
and Dobberstein, B. (1995) The proteinconducting channel in the membrane of the
endoplasmic reticulum is open laterally
toward the lipid bilayer. Cell, 81, 207-214.
Matlack, K., Mothes, W. and Rapoport, T.
(1998) Protein translocation: Tunnel vision.
Cell, 92, 381-390.
McCormick, P.J., Miao, Y., Shao, Y., Lin, J.
and Johnson, A.E. (2003) Cotranslational
protein integration into the ER membrane is
mediated by the binding of nascent chains to
translocon proteins. Mol Cell, 12, 329-341.
Loo, M.A., Jensen, T.J., Cui, L., Hou, Y.,
Chang, X.B. and Riordan, J.R. (1998)
Perturbation of Hsp90 interaction with nascent
CFTR prevents its maturation and accelerates
its degradation by the proteasome. EMBO J, 17,
6879-6887.
McGovern, K. and Beckwith, J. (1991)
Membrane insertion of the Escherichia coli MalF
protein in cells with impaired secretion
machinery. J Biol Chem, 266, 20870-20876.
Ma, C. and Chang, G. (2004) Structure of the
multidrug resistance efflux transporter EmrE
from Escherichia coli. Proc Natl Acad Sci U S A,
101, 2852-2857.
Meacham, G.C., Lu, Z., King, S., Sorscher, E.,
Tousson, A. and Cyr, D.M. (1999) The Hdj2/Hsc70 chaperone pair facilitates early steps
in CFTR biogenesis. EMBO J, 18, 1492-1505.
MacKenzie, K.R., Prestegard, J.H. and
Engelman, D.M. (1997) A transmembrane
helix dimer: structure and implications.
Science, 276, 131-133.
Meacock, S.L., Greenfield, J.J. and High, S.
(2000) Protein targeting and translocation at
the endoplasmic reticulum membrane-through the eye of a needle? Essays Biochem, 36,
1-13.
Manoil, C. (1991) Analysis of membrane
protein topology using alkaline phosphatase
and ß-galactosidase gene fusions. Methods in
Cell Biol, 34, 61-75.
Manoil, C. and Beckwith, J. (1986) A genetic
approach to analyzing membrane protein
topology. Science, 233, 1403-1408.
Meacock, S.L., Lecomte, F.J., Crawshaw, S.G.
and High, S. (2002) Different transmembrane
domains associate with distinct endoplasmic
reticulum components during membrane
integration of a polytopic protein. Mol Biol Cell,
13, 4114-4129.
Melén, K., Krogh, A. and von Heijne, G.
(2003) Reliability measures for membrane
47
protein topology prediction algorithms. J Mol
Biol, 327, 735-744.
Menetret, J.F., Hegde, R.S., Heinrich, S.U.,
Chandramouli, P., Ludtke, S.J., Rapoport,
T.A. and Akey, C.W. (2005) Architecture of the
ribosome-channel complex derived from
native membranes. J Mol Biol, 348, 445-457.
Menetret, J.F., Neuhof, A., Morgan, D.G.,
Plath, K., Radermacher, M., Rapoport, T.A.
and Akey, C.W. (2000) The structure of
ribosome-channel complexes engaged in
protein translocation. Mol Cell, 6, 1219-1232.
Meyer, T.H., Menetret, J.F., Breitling, R.,
Miller, K.R., Akey, C.W. and Rapoport, T.A.
(1999) The bacterial SecY/E translocation
complex forms channel-like structures similar
to those of the eukaryotic Sec61p complex. J
Mol Biol, 285, 1789-1800.
Monné, M., Hermansson, M. and von Heijne,
G. (1999a) A turn propensity scale for
transmembrane helices. J Mol Biol, 288, 141145.
Murata, K., Mitsuoka, K., Hirai, T., Walz, T.,
Agre, P., Heymann, J.B., Engel, A. and
Fujiyoshi, Y. (2000) Structural determinants of
water permeation through aquaporin-1.
Nature, 407, 599-605.
Möller, J. and Rydström, J. (1999) The
membrane topology of proton-pumping
Escherichia coli transhydrogenase determined
by cysteine labeling. J Biol Chem, 274, 1907219080.
Möller, S., Croning, M. and Apweiler, R.
(2001) Evaluations of methods for the
predictive evaluation of membrane spanning
regions. Bioinformatics, 17, 646-653.
Möller, S., Kriventseva, E. and Apweiler, R.
(2000) A collection of well-characterised
integral membrane proteins. Bioinformatics, 16,
1159-1160.
Nilsson, I., Johnson, A.E. and von Heijne, G.
(2003) How hydrophobic is alanine? J Biol
Chem, 278, 29389-29393.
Monné, M., Nilsson, I., Elofsson, A. and von
Heijne, G. (1999b) Turns in transmembrane
helices: Determination of the minimal length of
a ''helical hairpin'' and derivation of a finegrained turn propensity scale. J Mol Biol, 293,
807-814.
Nilsson, I., Sääf, A., Whitley, P., Gafvelin, G.,
Waller, C. and von Heijne, G. (1998) Prolineinduced disruption of a transmembrane αhelix in its natural environment. J Mol Biol, 284,
1165-1175.
Mothes, W., Heinrich, S., Graf, R., Nilsson, I.,
von Heijne, G., Brunner, J. and Rapoport, T.
(1997) Molecular mechanisms of membrane
protein integration into the endoplasmic
reticulum. Cell, 89, 523-533.
Nilsson, I. and von Heijne, G. (1993)
Determination of the distance between the
oligosaccharyltransferase active site and the
endoplasmic reticulum membrane. J Biol Chem,
268, 5798-5801.
Mothes, W., Jungnickel, B., Brunner, J. and
Rapoport, T.A. (1998) Signal sequence
recognition in cotranslational translocation by
protein components of the endoplasmic
reticulum membrane. J Cell Biol, 142, 355-364.
Nilsson, I.M. and von Heijne, G. (1990) Finetuning the Topology of a Polytopic Membrane
Protein. Role of Positively and Negatively
Charged Residues. Cell, 62, 1135-1141.
Mothes, W., Prehn, S. and Rapoport, T.A.
(1994) Systematic probing of the environment
of a translocating secretory protein during
translocation through the ER membrane.
EMBO J, 13, 3937-3982.
Nilsson, J., Persson, B. and von Heijne, G.
(2005) Comparative analysis of amino acid
distributions in integral membrane proteins
from 107 genomes. Proteins, 60, 606-616.
48 Marika Cassel
Oliver, J., Jungnickel, B., Görlich, D.,
Rapoport, T. and High, S. (1995) The Sec61
complex is essential for the insertion of
proteins into the membrane of the
endoplasmic reticulum. FEBS Lett, 362, 126130.
Opella, S.J. (1997) NMR and membrane
proteins. Nature Struct Biology, 4, 845-848.
Orlandini, E., Seno, F., Banavar, J.R., Laio, A.
and Maritan, A. (2000) Deciphering the
folding kinetics of transmembrane helical
proteins. Proc Natl Acad Sci U S A, 97, 1422914234.
Paetzel, M., Dalbey, R.E. and Strynadka,
N.C.J. (1998) Crystal structure of a bacterial
signal peptidase in complex with a β-lactam
inhibitor. Nature, 396, 186-190.
Palade, G. (1975) Intracellular aspects of the
process of protein synthesis. Science, 189, 347358.
Pervushin, K., Riek, R., Wider, G. and
Wuthrich, K. (1997) Attenuated T2 relaxation
by mutual cancellation of dipole-dipole
coupling and chemical shift anisotropy
indicates an avenue to NMR structures of very
large biological macromolecules in solution.
Proc Natl Acad Sci USA, 94, 12366-12371.
Pool, M.R., Stumm, J., Fulga, T.A., Sinning, I.
and Dobberstein, B. (2002) Distinct modes of
signal recognition particle interaction with the
ribosome. Science, 297, 1345-1348.
Popot, J.L. and Engelman, D.M. (1990)
Membrane protein folding and
oligomerization - The 2-stage model.
Biochemistry, 29, 4031-4037.
Prinz, W.A. and Beckwith, J. (1994) Gene
fusion analysis of membrane protein topology:
A direct comparison of alkaline phosphatase
and ß-lactamase fusions. J Bacteriol, 176, 64106413.
Rapoport, T.A., Goder, V., Heinrich, S.U. and
Matlack, K.E. (2004) Membrane-protein
integration and the role of the translocation
channel. Trends Cell Biol, 14, 568-575.
Rapoport, T.A., Jungnickel, B. and Kutay, U.
(1996) Protein transport across the eukaryotic
endoplasmic reticulum and bacterial inner
membranes. Annu Rev Biochem, 65, 271-303.
Rapp, M., Drew, D.E., Daley, D.O., Nilsson,
J., Carvalho, T., Melén, K., de Gier, J.W. and
von Heijne, G. (2004) Experimentally based
topology models for E. coli inner membrane
proteins. Prot Sci, 13, 937-945.
Roberts, C.H. and Chlebowski, J.F. (1984)
"PhoA+T X-100+trypsin". J Biol Chem, 259, 729733.
Rost, B., Fariselli, P. and Casadio, R. (1996)
Topology prediction for helical
transmembrane proteins at 86% accuracy. Prot
Sci, 5, 1704-1718.
Sadlish, H., Pitonzo, D., Johnson, A.E. and
Skach, W.R. (2005) Sequential triage of
transmembrane segments by Sec61α during
biogenesis of a native multispanning
membrane protein. Nat Struct Mol Biol, 12,870878.
Sato, M., Hresko, R. and Mueckler, M. (1998)
Testing the charge difference hypothesis for
the assembly of a eucaryotic multispanning
membrane protein. J Biol Chem, 273, 2520325208.
Sato, M. and Mueckler, M. (1999) A conserved
amino acid motif (R-X-G-R-R) in the GLUT1
glucose transporter is an important
determinant of membrane topology. J Biol
Chem, 274, 24721-24725.
Schatz, G. and Dobberstein, B. (1996)
Common principles of protein translocation
across membranes. Science, 271, 1519-1526.
Scheuring, S., Muller, D.J., Stahlberg, H.,
Engel, H.A. and Engel, A. (2002) Sampling the
conformational space of membrane protein
surfaces with the AFM. Eur Biophys J, 31, 172178.
49
Schiffer, M., Chang, C.H. and Stevens, F.J.
(1992) The Functions of Tryptophan Residues
in Membrane Proteins. Prot Eng, 5, 213-214.
Schirmer, T., Keller, T. A., Wang, Y. F. and
Rosenbusch, J. P. (1995) Structural basis for
sugar translocation through maltoporin
channels at 3.1 Ångstrom resolution Science,
267, 512-514
Schulz, G.E. (2000) β-Barrel membrane
proteins. Curr Opin Struct Biol, 10, 443-447.
Senes, A., Ubarretxena-Belandia, I. and
Engelman, D.M. (2001) The Calpha ---H...O
hydrogen bond: a determinant of stability and
specificity in transmembrane helix
interactions. Proc Natl Acad Sci U S A, 98, 90569061.
Simon, S.M. and Blobel, G. (1991) A ProteinConducting Channel in the Endoplasmic
Reticulum. Cell, 65, 371-380.
Simons, K. and Ikonen, E. (1997) Functional
rafts in cell membranes. Nature, 387, 569-572.
Simons, K. and Ikonen, E. (2000) How cells
handle cholesterol. Science, 290, 1721-1726.
Singer, S.J. and Nicolson, G.L. (1972) The
fluid mosaic model of the structure of cell
membranes. Science, 175, 720-731.
Song, L., Hobaugh, M.R., Shustak, C.,
Ceheley, S., Bayley, H. and Gouaux, J.E.
(1996) Structure of Staphylococcal αhemolysin, a heptameric transmembrane pore.
Science, 274, 1859-1866.
Sonnhammer, E., von Heijne, G. and Krogh,
A. (1998) A hidden Markov model for
predicting transmembrane helices in protein
sequences. Intell Syst Mol Biol, 6, 175-182.
Stroud, R. (1995) Ion channel forming colicins.
Curr Opin Struct Biol, 5, 514-520.
Subramaniam, S. and Henderson, R. (1999)
Electron crystallography of bacteriorhodopsin
with millisecond time resolution. J Struct Biol,
128, 19-25.
Swanton, E., High, S. and Woodman, P. (2003)
Role of calnexin in the glycan-independent
quality control of proteolipid protein. EMBO J,
22, 2948-2958.
Sääf, A., Johansson, M., Wallin, E. and von
Heijne, G. (1999) Divergent evolution of
membrane protein topology: The Escherichia
coli RnfA and RnfE homologues. Proc Natl Acad
Sci USA, 96, 8540-8544.
Tadayyon, M., Zhang, Y., Gnaneshan, S.,
Hunt, L., Mehraein-Ghomi, F. and BroomeSmith, J.K. (1992) beta-Lactamase fusion
analysis of membrane protein assembly.
Biochem Soc Trans, 20, 598-601.
Tamm, L.K., Abildgaard, F., Arora, A., Blad,
H. and Bushweller, J.H. (2003) Structure,
dynamics and function of the outer membrane
protein A (OmpA) and influenza
hemagglutinin fusion domain in detergent
micelles by solution NMR. FEBS Lett, 555, 139143.
Tamm, L.K., Arora, A. and Kleinschmidt, J.H.
(2001) Structure and assembly of beta-barrel
membrane proteins. J Biol Chem, 276, 3239932402.
Tenson, T. and Ehrenberg, M. (2002)
Regulatory nascent peptides in the ribosomal
tunnel. Cell, 108, 591-594.
Thrift, R.N., Andrews, D.W., Walter, P. and
Johnson, A.E. (1991) A Nascent Membrane
Protein Is Located Adjacent to ER Membrane
Proteins Throughout Its Integration and
Translation. J Cell Biol, 112, 809-821.
Traxler, B., Boyd, D. and Beckwith, J. (1993)
The topological analysis of integral
cytoplasmic membrane proteins. J Membr Biol,
132, 1-11.
Tusnady, G.E. and Simon, I. (1998) Principles
governing amino acid composition of integral
50 Marika Cassel
membrane proteins: Application to topology
prediction. J Mol Biol, 283, 489-506.
Tusnady, G.E. and Simon, I. (2001) The
HMMTOP transmembrane topology
prediction server. Bioinformatics, 17, 849-850.
Weiss, M.S., Abele, U., Weckesser, J., Welte,
W., Schiltz, E. and Schulz, G.E. (1991)
Molecular architecture and electrostatic
properties of a bacterial porin. Science, 254,
1627-1630.
Ubarretxena-Belandia, I., Baldwin, J.M.,
Schuldiner, S. and Tate, C.G. (2003) Threedimensional structure of the bacterial
multidrug transporter EmrE shows it is an
asymmetric homodimer. EMBO J, 22, 61756181.
Vereb, G., Szollosi, J., Matko, J., Nagy, P.,
Farkas, T., Vigh, L., Matyus, L., Waldmann,
T.A. and Damjanovich, S. (2003) Dynamic, yet
structured: The cell membrane three decades
after the Singer-Nicolson model. Proc Natl Acad
Sci U S A, 100, 8053-8058.
Ubarretxena-Belandia, I. and Tate, C.G. (2004)
New insights into the structure and oligomeric
state of the bacterial multidrug transporter
EmrE: an unusual asymmetric homo-dimer.
FEBS Lett, 564, 234-238.
Wessels, H.P. and Spiess, M. (1988) Insertion
of a multispanning membrane protein occurs
sequentially and requires only one signal
sequence. Cell, 55, 61-70.
Wahlberg, J.M. and Spiess, M. (1997) Multiple
determinants direct the orientation of signalanchor proteins: the topogenic role of the
hydrophobic signal domain. J Cell Biol, 137,
555-562.
Wallin, E., Tsukihara, T., Yoshikawa, S., von
Heijne, G. and Elofsson, A. (1997)
Architecture of helix bundle membrane
proteins: An analysis of cytochrome c oxidase
from bovine mitochondria. Protein Sci, 6, 808815.
Wallin, E. and von Heijne, G. (1998) Genomewide analysis of integral membrane proteins
from eubacterial, archaean, and eukaryotic
organisms. Protein Sci, 7, 1029-1038.
Walter, P. and Johnson, A.E. (1994) Signal
sequence recognition and protein targeting to
the endoplasmic reticulum membrane. Annu
Rev Cell Biol, 10, 87-119.
Van den Berg, B., Clemons, W.M., Jr.,
Collinson, I., Modis, Y., Hartmann, E.,
Harrison, S.C. and Rapoport, T.A. (2004) Xray structure of a protein-conducting channel.
Nature, 427, 36-44.
van Meer, G. (1989) Lipid traffic in animal
cells. Annu Rev Cell Biol, 5, 247-275.
Whitley, P., Grahn, E., Kutay, U., Rapoport, T.
and von Heijne, G. (1996) A 12 residues long
poly-leucine tail is sufficient to anchor
syntaptobrevin to the ER membrane. J Biol
Chem, 271, 7583-7586.
Wolfe, P.B., Wickner, W. and Goodman, J.M.
(1983) Sequence of the leader peptidase gene of
Escherichia coli and the orientation of leader
peptidase in the bacterial envelope. J Biol
Chem, 258, 12073-12080.
von Heijne, G. (1986) The distribution of
positively charged residues in bacterial inner
membrane proteins correlates with the transmembrane topology. EMBO J, 5, 3021-3027.
von Heijne, G. (1989) Control of topology and
mode of assembly of a polytopic membrane
protein by positively charged residues. Nature,
341, 456-458.
von Heijne, G. (1990) The signal peptide. J
Membr Biol, 115, 195-201.
von Heijne, G. (1991) Proline kinks in
transmembrane α-helices. J Mol Biol, 218, 499503.
von Heijne, G. (1992) Membrane protein
structure prediction - Hydrophobicity analysis
51
and the positive-inside rule. J Mol Biol, 225,
487-494.
secretory proteins differ in FRET-detected
folding. Cell, 116, 725-736.
von Heijne, G. (1994) Membrane proteins:
From sequence to structure. Annu Rev Biophys
Biomol Struct, 23, 167-192.
Zhou, F.X., Cocco, M.J., Russ, W.P., Brunger,
A.T. and Engelman, D.M. (2000) Interhelical
hydrogen bonding drives strong interactions
in membrane proteins. Nature Struct Biol, 7,
154-160.
von Heijne, G. (1996) Principles of membrane
protein assembly and structure. Progr Biophys
Mol Biol, 66, 113-139.
von Heijne, G. (1997) Getting greasy: How
transmembrane polypeptide segments
integrate into the lipid bilayer. Mol Microbiol,
24, 249-253.
von Heijne, G. and Gavel, Y. (1988) Topogenic
signals in integral membrane proteins. Eur J
Biochem, 174, 671-678.
Woolhead, C.A., McCormick, P.J. and
Johnson, A.E. (2004) Nascent membrane and
Zhou, F.X., Merianos, H.J., Brunger, A.T. and
Engelman, D.M. (2001) Polar residues drive
association of polyleucine transmembrane
helices. Proc Natl Acad Sci USA, 98, 2250-2255.
Zopf, D., Bernstein, H.D., Johnson, A.E. and
Walter, P. (1990) The Methionine-Rich Domain
of the 54 kd Protein Subunit of the Signal
Recognition Particle Contains an RNA Binding
Site and Can Be Crosslinked to a Signal
Sequence. EMBO J, 9, 4511-4517.
I
Article No. jmbi.1999.2657 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 288, 141–145
A Turn Propensity Scale for Transmembrane Helices
Magnus MonneÂ, Marika Hermansson and Gunnar von Heijne*
Department of Biochemistry
Stockholm University
S-106 91 Stockholm, Sweden
Using a model protein with a 40 residue hydrophobic transmembrane
segment, we have measured the ability of all the 20 naturally occurring
amino acids to form a tight turn when placed in the middle of the hydrophobic segment. Turn propensities in a transmembrane helix are found
to be markedly different from those of globular proteins, and in most
cases correlate closely with the hydrophobicity of the residue. The turn
propensity scale may be used to improve current methods for membrane
protein topology prediction.
# 1999 Academic Press
*Corresponding author
Keywords: membrane protein; turn propensity; secondary structure;
transmembrane helix
Introduction
The formation of tight turns in globular proteins
has been studied for decades, both experimentally
and by statistical analysis of known structures, and
reliable turn propensity scales have been established (Creighton, 1993; von Heijne, 1987). Remarkably, however, essentially nothing is known about
the residue characteristics responsible for the formation of tight turns between transmembrane
a-helices in integral membrane proteins. This is
due in part to the paucity of high-resolution structural information for this class of proteins, but it is
nevertheless surprising that no direct means of
measuring turn propensities in membrane proteins
has been established.
We recently developed a simple experimental
system for the analysis of turn formation in transmembrane helices embedded in the membrane of
the endoplasmic reticulum (Nilsson & von Heijne,
1998), and could show that a single proline residue
placed near the middle of a 40 residue poly(Leu)
transmembrane helix ef®ciently converts the
poly(Leu) segment from a single, long transmembrane helix to a tightly spaced pair of
transmembrane helices (a ``helical hairpin''). We
have now used this system to measure the turn
propensities for all the 20 naturally occurring
amino acid residues, and present the ®rst experimental propensity scale for the formation of tight
turns between transmembrane a-helices. This scale
is shown to differ in important respects from turn
propensities in globular proteins, and in general
Abbreviations used: BSA, bovine serum albumin.
E-mail address of the corresponding author:
[email protected]
0022-2836/99/160141±05 $30.00/0
correlates better with residue hydrophobicity than
with ``classical'' secondary structure propensities.
Results
A system for measuring turn propensities in
transmembrane helices under in vivolike conditions
For these studies, we have used the well-characterized Escherichia coli protein Lep, which contains
two transmembrane helices (H1 and H2) and a
large C-terminal domain (P2). When expressed
in vitro in the presence of dog pancreas microsomes, Lep has been shown to insert into the
microsomal membrane with both the N and C termini on the luminal side (Nilsson & von Heijne,
1993), i.e. in the same orientation as it normally
inserts into the inner membrane of E. coli (Wolfe
et al., 1983). Translocation of the P2 domain to the
lumenal side is conveniently assayed by the glycosylation of a unique acceptor site for N-linked glycosylation (Asn-Ser-Thr) placed 20 residues
downstream of H2 (Figure 1(a)). An advantage of
this approach is that the microsomal in vitro system
closely mimics the conditions of in vivo membrane
protein assembly into the endoplasmic reticulum
membrane.
For the studies reported here, H2 was replaced
by a 40 residue poly(Leu) segment (including one
Val) ¯anked by four lysine residues on the N-terminal end and by a Gln-Gln-Gln-Pro stretch on the
C-terminal end. Given that typical transmembrane
helices are 20-30 residues long (Bowie, 1997), this
poly(Leu) stretch should, in principle, be able to
form either one long or two closely spaced transmembrane helices. Indeed, the poly(Leu) segment
has previously been shown to insert into the micro# 1999 Academic Press
142
Membrane Protein Turn Propensities
somal membrane as a single transmembrane helix
with the P1 loop in the cytoplasm and the P2
domain in the microsomal lumen, and it was
observed that the introduction of a Pro residue
near the middle of the poly(Leu) stretch results in
the formation of a helical hairpin in the membrane
and localization of the P2 domain to the cytoplasmic side (Nilsson & von Heijne, 1998).
Using the same poly(Leu) construct, we have
now measured turn propensities for all the 20
naturally occurring amino acids by substituting
each residue (X) for Leu22 near the middle of the
poly(Leu) segment, and expressing the L22 ! X
constructs in vitro in the presence of dog pancreas
microsomes (Figure 1(b)). Based on the quanti®cation of the glycosylation ef®ciencies given in
Figure 1(c), a scale of turn propensities can be
derived from this set of data (Table 1).
To rule out that lack of glycosylation is the result
of inef®cient insertion of the poly(Leu) stretch into
the microsomal membrane rather than formation
of a helical hairpin structure, a segment encompassing H1 and part of the P1 domain (residues
5-46) was deleted from two poorly glycosylated
constructs (L22 ! R and L22 ! E), and membrane
insertion of the L22 ! R(5-46) and L22 !
E(5-46) constructs was monitored by alkaline
extraction of the microsomes. This treatment is
known to remove peripherally bound membrane
proteins, but leaves properly inserted transmembrane proteins in the membrane pellet (Fujiki et al.,
1982). In addition, the P2 domain (residues 79-323)
was expressed alone to make sure that any membrane association observed for the 5-46 constructs
was due only to the poly(Leu) segment. As seen in
Figure 2, the two 5-46 constructs remained with
the membrane pellet, whereas the P2 domain,
Table 1. Turn propensities for amino acid residues in a
transmembrane helix
Residue
Figure 1. (a) Model protein used in this study. The
H2 transmembrane segment in Lep was replaced with
a stretch of residues of the general design
LIK4L21XL7VL10Q3P, where X is one of the 20 naturally
occurring amino acid residues. A glycosylation acceptor
site was placed 20 residues downstream of H2 (counting
from the ®rst Gln residue after the hydrophobic stretch).
Depending on the luminal or cytoplasmic localization of
the P2 domain, the glycosylation acceptor site will either
be modi®ed (Y) or not ( ). Note that the tilted conformation of the model single-spanning transmembrane H2
helix
has
not
been
experimentally
proven.
(b) The indicated L22 ! X mutants were translated
in vitro in the presence of rough microsomes and analyzed by SDS-PAGE. Black and white dots indicate the
glycosylated and non-glycosylated forms of the proteins,
respectively. (c) Quanti®cation of the gels shown in
(b). The percentage glycosylation was calculated as 100
I‡/(I‡ ‡ Iÿ), where I‡ (Iÿ) is the intensity of the glycosylated (non-glycosylated) band. From duplicate experiments on all the 20 constructs, the typical error in the
determination of glycosylation ef®ciency was 45 %
(bars), except for W where the error was 10 %.
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
Turn propensity
0.5
0.6
1.6
1.6
0.4
1.3
1.6
0.6
1.6
0.4
0.5
1.7
1.7
1.6
1.7
0.7
0.4
0.5
0.7
0.6
The turn propensity is de®ned as (1 ÿ fX)/m(1 ÿ fX), where fX
is the fraction of glycosylated molecules in the L22 ! X mutant
and m(1 ÿ fX) is the mean value of 1 ÿ fX over all 20 residues.
The typical error in the propensity values is 0.05.
Membrane Protein Turn Propensities
143
Figure 2. Alkaline extraction of
the L22 ! R(5-46) (lanes 2, 5, 8),
L22 ! E(5-46) (lanes 3, 6, 9),
and P2 (lanes 4, 7, 10) constructs.
Constructs were translated in vitro
in the absence (lanes 2-4) or presence (lanes 5-10) of rough microsomes. In lanes 5-10, microsomes
were subjected to a sodium carbonate wash before loading onto
the gel. p, pellet; s, supernatant.
Black and white dots indicate glycosylated and non-glycosylated
forms of the proteins, respectively.
The 5-46 constructs lack H1 and
about two-thirds of the P1
domain, and the P2 construct
lacks residues 2-78, i.e. the entire
H1-P1-H2 domain. Lane 1 contains molecular mass markers as
indicated.
when expressed alone, was found exclusively in
the supernatant. We conclude that the degree of
glycosylation seen for the different L22 ! X
mutants accurately re¯ects the fraction of molecules that insert with a single transmembrane segment versus a helical hairpin, and that it can thus
be used as a basis for the turn propensity scale presented in Table 1.
Discussion
We have used a simple in vivo-like system where
the membrane topology adopted by a 40 residue
long model transmembrane segment can be used
to directly infer a turn propensity scale relevant for
transmembrane a-helices. As seen in Figure 1(c),
hydrophobic residues (L, F, A, Y, V, and I) do not
induce a turn in the poly(Leu) helix, whereas
charged or highly polar residues do. In addition,
the two classical helix breakers Pro and Gly both
induce a turn (Pro somewhat more ef®ciently than
Gly). Interestingly, despite their polar nature, Ser
and Thr do not have high turn propensities. It is
known from helices in globular proteins that Ser,
Thr, and Cys side-chains can form hydrogen bonds
to the polypeptide backbone (Gray & Matthews,
1984), which might increase their apparent hydrophobicity when present in a transmembrane helix,
making turn formation less favorable. Consistent
with this, Ser and Thr are rather frequently found
in transmembrane helices, in contrast to Asn and
Gln (von Heijne, 1992). Finally, Trp is known to
have the strongest preference for the lipid-water
interface of all the amino acid residues (Wimley &
White, 1996), which may explain its somewhat
higher turn propensity compared to, e.g. Phe and
Tyr. Although the data reported in Figure 1(c) and
Table 1 are largely consistent with a two-tier system where residues have either a high or a low
turn propensity, it may be possible to provide a
better discrimination between different residues in
the transition region between high and low turn
propensity by inserting pairs of residues in the
middle of the model transmembrane segment; such
studies are in progress.
The turn propensity scale derived here deviates
signi®cantly from the turn propensities observed in
globular proteins (Figure 3, top panel). Thus, while
the charged and highly polar amino acids all have
high turn propensities in the transmembrane helix
context, this is not the case in globular proteins.
Ser, in contrast, has a rather high turn propensity
in globular proteins but not in transmembrane
helices. Pro and Gly are turn promoters in both
contexts. The correlation between the turn propensity scale and the so-called interface hydrophobicity scale (Wimley & White, 1996) is not very
strong (Figure 3, middle panel), whereas the correlation with helical propensities in n-butanol (Liu &
Deber, 1998) is somewhat better (Figure 3, bottom
panel). Good correlations are also obtained with
some statistically de®ned hydrophobicity scales
(von Heijne, 1992; results not shown).
In summary, we have measured the turn propensities for all the 20 naturally occurring amino
acids placed in the middle of a poly(Leu) segment
that is long enough to form either a single or two
closely spaced transmembrane helices. Most of our
results can be explained by hydrophobicity: all
hydrophobic residues prefer the membrane
environment over the membrane-water interface
region (Wimley & White, 1996), and have low turn
propensities. Conversely, the charged and highly
polar residues induce turn formation in order to
avoid the membrane interior. Thus, intrinsic conformational preferences become largely irrelevant
in the context of a transmembrane helix, as
observed previously in peptide studies of a versus
b-structure formation in water, detergent, and lipid
vesicle environments (Deber & Li, 1995; Li &
Deber, 1994). In the context of the microsomal
membrane, Pro behaves as a strongly polar residue, presumably because its inclusion in a transmembrane helix necessitates the disruption of at
144
least one hydrogen bond. Perhaps the most surprising result is that Gly has such a high turn propensity, since it neither has a polar side-chain, nor
disrupts backbone hydrogen bonds when in a
Membrane Protein Turn Propensities
helix. Apparently, its exceptional conformation
¯exibility suf®ces to make the turn conformation
preferred over the intact transmembrane helix. A
possible mechanism for turn formation in a transmembrane helix during its insertion into the membrane of the endoplasmic reticulum has been
suggested previously (Nilsson & von Heijne, 1998).
Finally, we anticipate that the turn propensity
scale presented here will improve our ability to
distinguish between cases of a single long and two
closely spaced transmembrane helices when predicting membrane protein topology from amino
acid sequence information.
Materials and Methods
Enzymes and chemicals
Unless otherwise stated, all enzymes were from Promega. T7 DNA polymerase, [35S]Met, ribonucleotides,
deoxyribonucleotides, dideoxyribonucleotides, and the
cap analog m7G(50 )ppp(50 )G were from Amersham-Pharmacia (Uppsala, Sweden). Plasmid pGEM1, DTT, bovine
serum albumin (BSA), SP6 RNA polymerase, RNasin
and rabbit reticulocyte lysate were from Promega. Spermidine was from Sigma. Oligonucleotides were from
Cybergene (Stockholm, Sweden).
DNA manipulations
Figure 3. Turn propensities in transmembrane helices
are different from turn propensities in globular proteins.
Top, the turn propensity scale from Table 1 is plotted
against a typical turn propensity scale for globular proteins (Williams et al., 1987); middle, the interface hydrophobicity scale described by Wimley & White (1996);
bottom, a scale of helical propensities in n-butanol (Liu
& Deber, 1998). Correlation coef®cients are indicated in
the respective panels.
For cloning into and expression from the pGEM1 plasmid, the 50 end of the lep gene was modi®ed: ®rst, by the
introduction of an XbaI site and; second, by changing the
context 50 to the initiator ATG codon to a ``Kozak consensus'' sequence (Johansson et al., 1993; Kozak, 1989).
Replacement of the H2 region in Lep was performed by
®rst introducing BclI and NdeI restriction sites in codons
59 and 80 ¯anking the H2 region, and then replacing
the BclI-NdeI fragment by the appropriate doublestranded oligonucleotides. Residues 59-80 in H2 were
replaced by poly(Leu) sequences of the general design
LIK4L21XL7VL10Q3P, where X is one of the 20 naturally
occurring amino acids; the Val residue results from the
inclusion of a SpeI restriction site. The (5-46) and P2
constructs were made by deleting, respectively, residues
5-46 and 2-78 in Lep. Site-speci®c mutagenesis used to
add BclI and NdeI restriction sites at the 30 and 50 ends of
H2 in Lep and to introduce Asn-Ser-Thr acceptor sites
for N-linked glycosylation was performed according to
the Kunkel method (Geisselsoder et al., 1987; Kunkel,
1987) . Glycosylation acceptor sites were designed as
described (Nilsson et al., 1994), i.e. by replacing three
appropriately positioned codons with codons for the
acceptor tripeptide Asn-Ser-Thr. For the L22 ! X substitutions, the QuickChange2 site-directed mutagenesis kit
from Stratagene was used. Some of the primers were
designed with a degenerate base in the second position
of the codon for X in order to get more than one mutant
from the primer pair. All mutants were con®rmed by
DNA sequencing of plasmid using T7 DNA polymerase.
Expression in vitro
The constructs in pGEM1 were transcribed by SP6
RNA polymerase for one hour at 37 C. The transcription
mixture was as follows: 1-5 mg DNA template, 5 ml of
10 SP6 H-buffer (400 mM Hepes-KOH (pH 7.4),
Membrane Protein Turn Propensities
60 mM Mg acetate, 20 mM spermidine-HCl), 5 ml of
1 mg/ml BSA, 5 ml of 10 mM m7G(50 )ppp(50 )G, 5 ml of
50 mM DTT, 5 ml of rNTP mix (10 mM ATP, 10 mM
CTP, 10 mM UTP, 5 mM GTP), 18.5 ml of H2O, 1.5 ml of
RNase inhibitor (50 units), 0.5 ml of SP6 RNA polymerase
(20 units). Translation was performed in reticulocyte
lysate in the presence and absence of dog pancreas
microsomes (LiljestroÈm & Garoff, 1991) . Sodium carbonate extraction of microsomes was carried out as
described (Sakaguchi et al., 1987) . Translation products
were analyzed by SDS-PAGE and gels were quanti®ed
on a Fuji BAS1000 phosphoimager using the MacBAS
2.31 software. The glycosylation ef®ciency of a given
mutant was calculated as the quotient between the intensity of the glycosylated band divided by the summed
intensities of the glycosylated and non-glycosylated
bands.
Acknowledgments
This work was supported by grants from the Swedish
Cancer Foundation, the Swedish Natural and Technical
Sciences Research Councils, and the GoÈran Gustafsson
Foundation to G.v.H.
References
Bowie, J. U. (1997). Helix packing in membrane proteins.
J. Mol. Biol. 272, 780-789.
Creighton, C. (1993). Proteins: Structures and Molecular
Properties, 2nd edit., W. H. Freeman & Co., New
York.
Deber, C. M. & Li, S. C. (1995). Peptides in membranes:
helicity and hydrophobicity. Biopolymers, 37, 295318.
Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B.
(1982). Isolation of intracellular membranes by
means of sodium carbonate treatment. J. Cell Biol.
93, 97-102.
Geisselsoder, J., Witney, F. & Yuckenberg, P. (1987). Ef®cient site-directed in vitro mutagenesis. BioTechniques, 5, 786-791.
Gray, T. M. & Matthews, B. W. (1984). Intrahelical
hydrogen bonding of serine, threonine and cysteine
residues within alpha-helices and its relevance to
membrane-bound proteins. J. Mol. Biol. 175, 75-81.
Johansson, M., Nilsson, I. & von Heijne, G. (1993). Positively charged amino acids placed next to a signal
sequence block protein translocation more ef®-
145
ciently in Escherichia coli than in mammalian microsomes. Mol. Gen. Genet. 239, 251-256.
Kozak, M. (1989). Context effects and inef®cient
initiation at non-AUG codons in eucaryotic cell-free
translation systems. Mol. Cell. Biol. 9, 5073-5080.
Kunkel, T. A. (1987). Rapid and ef®cient site-speci®c
mutagenesis without phenotypic selection. Methods
Enzymol. 154, 367-382.
Li, S.-C. & Deber, C. M. (1994). A measure of helical
propensity for amino acids in membrane environments. Nature Struct. Biol. 1, 368-373.
LiljestroÈm, P. & Garoff, H. (1991). Internally located
cleavable signal sequences direct the formation of
Semliki Forest virus membrane proteins from a
polyprotein precursor. J. Virol. 65, 147-154.
Liu, L. P. & Deber, C. M. (1998). Uncoupling hydrophobicity and helicity in transmembrane segments. aHelical propensities of the amino acids in non-polar
environments. J. Biol. Chem. 273, 23645-23648.
Nilsson, I. & von Heijne, G. (1993). Determination of the
distance between the oligosaccharyltransferase
active site and the endoplasmic reticulum membrane. J. Biol. Chem. 268, 5798-5801.
Nilsson, I. & von Heijne, G. (1998). Breaking the camel's
back: proline-induced turns in a model transmembrane helix. J. Mol. Biol. 284, 1185-1189.
Nilsson, I., Whitley, P. & von Heijne, G. (1994). The
C-terminal ends of internal signal and signal-anchor
sequences are positioned differently in the ER translocase. J. Cell Biol. 126, 1127-1132.
Sakaguchi, M., Mihara, K. & Sato, R. (1987). A short
amino-terminal segment of microsomal cytochrome
P-450 functions both as an insertion signal and as a
stop-transfer sequence. EMBO J. 6, 2425-2431.
von Heijne, G. (1987). Sequence Analysis in Molecular
Biology: Treasure Trove or Trivial Pursuit?, Academic
Press, San Diego.
von Heijne, G. (1992). Membrane protein structure prediction - hydrophobicity analysis and the positiveinside rule. J. Mol. Biol. 225, 487-494.
Williams, R., Chang, A., Juretic, D. & Loughran, S.
(1987). Secondary structure predictions and medium
range interactions. Biochim. Biophys. Acta, 916, 200204.
Wimley, W. C. & White, S. H. (1996). Experimentally
determined hydrophobicity scale for proteins at
membrane interfaces. Nature Struct. Biol. 3, 842-848.
Wolfe, P. B., Wickner, W. & Goodman, J. M. (1983).
Sequence of the leader peptidase gene of Escherichia
coli and the orientation of leader peptidase in the
bacterial envelope. J. Biol. Chem. 258, 12073-12080.
Edited by F. E. Cohen
(Received 16 December 1998; received in revised form 22 February 1999; accepted 22 February 1999)
II
doi:10.1006/jmbi.2000.3933 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 301, 191–197
Formation of Cytoplasmic Turns between Two Closely
Spaced Transmembrane Helices During Membrane
Protein Integration into the ER Membrane
Annika SaÈaÈf, Marika Hermansson and Gunnar von Heijne*
Department of Biochemistry
Stockholm University, S-106 91
Stockholm, Sweden
The helical hairpin, two closely spaced transmembrane helices separated
by a short turn, is a recurring structural element in integral membrane
proteins, and may serve as a compact unit that inserts into the membrane
en bloc. Previously, we have determined the propensities of the 20 natural
amino acids, when present in the middle of a long hydrophobic stretch,
to induce the formation of a helical hairpin with a lumenally exposed
turn during membrane protein assembly into the endoplasmic reticulum
membrane. Here, we present results from a similar set of measurements,
but with the turn placed on the cytoplasmic side of the membrane. We
®nd that a signi®cantly higher number of turn-promoting residues need
to be present to induce a cytoplasmic turn compared to a lumenal turn,
and that, in contrast to the lumenal turn, the positively charged residues
Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These
results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.
# 2000 Academic Press
*Corresponding author
Keywords: membrane protein; topology; glycosylation; transmembrane
helix
Introduction
Transmembrane a-helices in integral membrane
proteins often form ``helical hairpins'', i.e. closely
spaced pairs of helices separated by a short turn.
In principle, the short turn could be located either
on the cytoplasmic or extra-cytoplasmic side of the
membrane, although the term ``helical hairpin''
was ®rst used to describe helices connected by an
extra-cytoplasmic turn (Engelman & Steitz, 1981).
Given the current models for co-translational insertion of transmembrane helices into the membrane
of the endoplasmic reticulum (ER) of eukaryotic
cells (Do et al., 1996; Hamman et al., 1998; Liao
et al., 1997; Mothes et al., 1997), it may well be that
the ``rules'' for forming helical hairpins with cytoplasmic and extra-cytoplasmic turns are different.
We recently measured the propensities of all the
20 naturally occurring amino acids to induce helical hairpin formation in a long hydrophobic
stretch, and were able to propose a turn propensity
scale for transmembrane helices (Monne et al.,
Abbreviations used: ER, endoplasmic reticulum; Lep,
leader peptidase.
E-mail address of the corresponding author:
[email protected]
0022-2836/00/010191±7 $35.00/0
1999a,b; Nilsson & von Heijne, 1998). In those studies, the turn was always placed on the lumenal
(extra-cytoplasmic) side of the ER membrane. We
now report similar measurements of turn propensities, but with the turn placed on the cytoplasmic
side of the membrane. We ®nd that the rank order
is similar to that found for lumenal turns, but with
weaker turn-promoting abilities. Thus, two or
more turn-promoting residues are needed to fully
induce a cytoplasmic turn, whereas a single residue with high turn propensity is enough to induce
a lumenal turn, suggesting that it may be generally
more dif®cult to induce the formation of helical
hairpins with cytoplasmic turns.
Results
A model system for measuring cytoplasmic
turn-propensities in transmembrane helices
inserted into the ER membrane
Leader peptidase (Lep) is a well-characterized
inner membrane protein from Escherichia coli containing two transmembrane helices (H1 and H2)
and a large C-terminal domain (P2) (Figure 1). Lep
inserts into the inner E. coli membrane and into
microsomes with the P1 loop facing the cytoplasm
# 2000 Academic Press
192
Cytoplasmic Turns in Membrane Proteins
Figure 1. Model proteins used in
this study. Wild-type Lep (I) has
two transmembrane segments (H1,
H2), a cytoplasmic loop (P1), and a
large extra-cytoplasmic domain
(P2). To study turn-induction in a
long hydrophobic transmembrane
segment (H3), a 40 residue long
poly(Leu) stretch was inserted in
the middle of the P2 domain, and
the glycosylation status of two
Asn-X-Thr glycosylation acceptor
sites ¯anking H3 was determined
after in vitro translation in the presence of dog pancreas microsomes
(glycosylated and non-glycosylated
glycosylation acceptor sites are
indicated by Y and Y, respectively). Constructs where H3 forms
a single transmembrane segment (II) will be glycosylated on only one site, whereas those where H3 forms a helical
hairpin (III) will be glycosylated on both sites. Turn-induction propensities were measured for all the 20 natural
amino acids by introducing them either singly or as a pair in the middle of H3 (arrows).
and the N terminus as well as the P2 domain in
the extra-cytoplasmic space (Johansson et al., 1993;
Wolfe et al., 1983). For the studies reported here, an
extra transmembrane segment (H3) composed of a
40 residue long poly(Leu) stretch of the composition . . . E225-T-S-(L)40-R-S-V233 . . . (the numbers
refer to residues in Lep) was introduced into the
periplasmic P2 domain (Figure 1). This poly(Leu)
stretch is long enough to form either a single transmembrane segment or a helical hairpin (MonneÂ
et al., 1999b). To study helical hairpin formation
in H3, one or a pair of each of the 20 natural
amino acids were introduced in the middle of the
poly(Leu) stretch, and their ability to induce a
cytoplasmic turn was assessed.
For the analysis of the constructs, we used a
standard in vitro translation system supplemented
with dog pancreas microsomes (LiljestroÈm &
Garoff, 1991). Since N-linked glycosylation is a
reliable marker for translocation of a domain to the
lumenal side of the microsomal membrane
(Nilsson & von Heijne, 1993), we introduced AsnX-Thr glycosylation acceptor sites in positions 96
and 258 ¯anking the poly(Leu) segment. As shown
in Figure 1, if H3 spans the membrane once only
one glycosylation-acceptor site (in position 96) will
be exposed to the lumen and glycosylated, whereas
if H3 forms a helical hairpin with a cytoplasmic
loop, both glycosylation acceptor sites will be
exposed to the lumenal side of the membrane and
hence glycosylated. Singly and doubly glycosylated molecules are easily distinguished by SDSPAGE.
As an example, results for a series of constructs
with zero to three proline residues replacing Leu20
in the middle of the H3 segment are shown in
Figure 2. Glycosylation of constructs with 40 leucine residues in H3 (lanes 1 and 2), with 1-3 proline residues in
the middle of H3 (lanes 3-5), and with a stretch of six lysine residues in the middle of H3 (lane 6). In lane 1, no
rough microsomes were added (-RM), whereas rough microsomes were present during translation in lanes 2-6. Molecules with zero, one, and two modi®ed glycosylation sites are indicated. The fraction of molecules with two modi®ed glycosylation sites is given below each lane.
Cytoplasmic Turns in Membrane Proteins
193
Figure 3. Ef®ciencies of cytoplasmic turn-induction for the 20
natural amino acids measured as
the fraction of molecules with two
modi®ed
glycosylation
sites
obtained after in vitro translation in
the presence of rough microsomes.
Results for constructs with a single
residue placed in the middle of H3
are shown by open bars, and for
constructs with a pair of residues
in the middle of H3 by ®lled bars.
As shown in the cartoons, formation of a cytoplasmic turn in H3
results in doubly glycosylated molecules. Arrows indicate the position
of the introduced residues.
Figure 2, together with a construct with six lysine
residues replacing Leu19-Leu22 in the middle of
H3 (6K) that serves as a positive control for a fully
induced, doubly glycosylated helical hairpin structure. A single Pro residue induces a helical hairpin
in only 36 % of the molecules, and three Pro residues are needed to reach the same level of doubly
glycosylated molecules, as seen for the 6K construct. This is in contrast to our previous studies of
lumenal turns, where one Pro residue was suf®cent
to induce full helical hairpin formation (Nilsson &
von Heijne, 1998).
Propensities of the 20 natural amino acids to
induce the formation of a helical hairpin with a
cytoplasmic loop
Lep-derived constructs with the 40 residue long
poly(Leu) H3 stretch or with one or a pair of each
of the natural amino acid placed in the middle of
H3, were expressed in vitro in the presence of dog
pancreas microsomes. The fraction of glycosylated
molecules with both acceptor sites modi®ed was
taken as a measure of turn propensity. For all constructs, the fraction of non-glycosylated molecules
was approximately the same (10-20 %), indicating
that targeting and membrane insertion per se was
not affected by the mutations in H3.
As shown in Figure 3 (open bars), none of the
singly inserted residues was able quantitatively to
promote the formation of a cytoplasmic turn,
although charged and polar amino acids all gave
some degree of helical hairpin formation. Pairs of
polar or charged residues had a higher turn-indu-
cing effect, though in most cases not reaching full
helical hairpin formation (®lled bars; note that the
maximum level of glycosylation reached in our
system is 80 %). Only the 2 K and 2R constructs
reached the same degree of helical hairpin formation as the 6 K construct. All the hydrophobic
residues plus Thr, Ser, Cys, Gly, Trp, and Tyr had
low or no turn-inducing effects even when present
in tandem.
A comparison with the previously measured
propensities to induce a lumenal turn (Monne et al.,
1999a,b) is shown in Figure 4. Since the lumenal
turn propensities were measured by placing the 40
residue long poly(Leu) stretch in the H2 position of
Lep (c.f. Figure 5), low levels of glycosylation of
the P2 domain correspond to ef®cient turn formation in this case. It is clear that turn propensities
for both single and pairs of polar and charged residues are generally lower on the cytoplasmic side
of the membrane. Moreover, although the rank
order of turn propensities are similar for lumenal
and cytoplasmic turns, the positively charged residues Arg and Lys have the highest cytoplasmic
turn propensities while Pro and Asn are the strongest turn-inducers on the lumenal side.
As a control to ensure that differences in the residues ¯anking the H2 segment used in our previous
studies and the H3 segment used in this study do
not compromise these comparisons, the H3 segment from the 1D, 1G, 1P, 1Q, 1S, and 1W mutants
(see Figure 3) plus their ¯anking regions were
introduced in place of a segment encompassing H2
and its ¯anking regions as shown in Figure 5 (top).
194
Cytoplasmic Turns in Membrane Proteins
Figure 4. Comparison between
ef®ciencies of helical hairpin formation with a lumenal turn (y-axis)
and a cytoplasmic turn (x-axis).
Lumenal turn propensities for
single (left panel) and pairs
(right panel) of residues were
measured (Monne et al., 1999a,b)
by placing a 40 residue long poly
(Leu) stretch with the compo
sitions
. . . K4L21XL7VL10QQQP . . .
or
. . . K4L20X2L7VL10QQQP . . .
(where X denotes one of the 20
natural amino acids) in the H2 position in Lep (c.f. Figure 5). Ef®cient
formation of a lumenal turn in H2
results in low levels of glycosylation (i.e. high percentage of nonglycosylated molecules), whereas
formation of a cytoplasmic turn in
H3 results in high levels of doubly glycosylated molecules, as indicated by the cartoons. The diagonal lines represent
equal ef®ciencies of lumenal and cytoplasmic turn formation (the maximum level of glycosylation in our system is
80-90 %). Note that not all pairs were tested in the H2 construct (right panel).
From Figure 4, we expected that these residues
should induce helical hairpin formation more ef®ciently on the lumenal side than on the cytoplasmic
side. Indeed, as shown in Figure 5 (bottom), all six
residues induced much higher degrees of helical
hairpin formation when the H3 region was transplanted into the H2 context. Thus, differences in
the regions ¯anking the poly(Leu) stretches in the
H2 and H3 contexts are not responsible for the
observed differences between lumenal and cytoplasmic turn-induction.
We conclude that, although the effects of the
different residues on helical hairpin formation on
the lumenal versus cytoplasmic side of the microsomal membrane are qualitatively similar, a signi®cantly smaller number of turn-promoting residues
are needed to induce a lumenal turn compared to
a cytoplasmic turn between two closely spaced
transmembrane helices.
Discussion
With the study reported here, we can now provide a rather complete picture of the turn-inducing
propensities of all the 20 natural amino acids when
present in the middle of a long hydrophobic transmembrane stretch inserted into the ER membrane.
Not surprisingly, hydrophobic amino acids do not
induce turns, charged residues are strong turn-promoters, and most polar residues are in-between.
Among the non-charged residues, Pro and Asn,
which are strong turn-promoters in globular proteins, have the highest turn propensities.
This general picture holds both for turns on the
cytoplasmic and lumenal sides of the ER membrane. However, there are some notable differences
between turn induction on the two sides. Thus, a
higher number of turn-promoting residues are
required to induce a turn on the cytoplasmic side
(Figure 4). Moreover, Arg and Lys are the two
strongest turn-promoting residues on the cytoplasmic side, but not on the lumenal side (where
Pro and Asn are stronger). Possibly, the relatively
high cytoplasmic turn propensities for Arg and Lys
is related to the ``positive inside'' rule, i.e. to the
general tendency of positively charged amino acids
to be enriched in cytoplasmic loops in integral
membrane proteins (von Heijne & Gavel, 1988;
Wallin & von Heijne, 1998).
These differences between cytoplasmic and
lumenal turns may be a re¯ection of the underlying membrane-insertion event. Current models
for membrane protein insertion into the ER generally posit that the transmembrane segments enter
the translocon complex one by one, with the ribosome bound on the top of the translocation channel
(Liao et al., 1997; Mothes et al., 1997). Within the
context of this model, the situation will thus be
different during the formation of a helical hairpin
with a lumenal loop compared to a helical hairpin
with a cytoplasmic loop (Figure 6). In the former
case, a hydrophobic stretch presumably goes
through a bent, helical hairpin-like state even if it
ends up as a single transmembrane segment,
whereas in the latter situation it will not bend at
all during the insertion process unless turn-promoting residues are present. It thus seems reasonable that helical hairpin formation will require a
smaller number of turn-promoting residues when
the turn is on the lumenal side.
Materials and Methods
Enzymes and chemicals
Unless otherwise stated, all enzymes were from Promega (Madison, WI, USA). T7 DNA polymerase, BclI,
195
Cytoplasmic Turns in Membrane Proteins
cyte lysate were from Promega. Oligonucleotides were
from Cybergene (Stockholm, Sweden).
DNA manipulations
Figure 5. Residues ¯anking the 40 residue long poly
(Leu) stretches in the model proteins used previously
for measuring lumenal turn propensities and in this
work for measuring cytoplasmic turn propensities are
not responsible for the observed differences in lumenal
versus cytoplasmic turn induction. For the experiments
shown, the entire region between a BclI site in lep codon
59 (three codons upstream of the poly(Leu) H2 segment)
in the 1P mutant (construct I) used by Monne et al.,
1999a and the C terminus was replaced by a region
from the 1D, 1G, 1P, 1Q, 1S, and 1W mutants constructed in this work (see Figures 1 and 2) starting 22
residues upstream of the H3 segment (construct II). As
shown in the lower panel, all six residues induced much
higher degrees of helical hairpin formation when the H3
region was transplanted into the H2 context than seen
in Figure 3. Note that helical hairpin formation in H2
results in high levels of non-glycosylated molecules
(open bars) whereas in H3 it corresponds to high levels
of doubly glycosylated molecules (®lled bars). The
arrows in the upper panel indicate the position of the
six residues in the two series of constructs.
[35S]Met, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and the cap analog m7G(50 )ppp(50 )G
were from Amersham-Pharmacia (Uppsala, Sweden).
Plasmid pGEM1, transcription buffer and rabbit reticulo-
All constructs were made from a recently described
Lep construct carried in the pING1 vector containing a
lep gene with SpeI and BglII restriction sites introduced
in codons 226-227 and 231-232 (SaÈaÈf et al., 1998). Introduction of the H3 region into the periplasmic P2 domain
of Lep was performed by replacing the SpeI-BglII fragment by a double-stranded oligonucleotide encoding a
stretch of 40 leucine residues ¯anked by SpeI and BamHI
(compatible with BglII) restriction sites, yielding an H3
region with the sequence . . . E225-T-S-(L)40-R-S-V233 . . .
(numbers refer to the wild-type Lep sequence). The lep
gene was further modi®ed by an Asn214 ! Gln
mutation (to remove a potential glycosylation site present in the wild-type Lep sequence) and by mutations
converting residues 96-98 and 258-260 to, respectively,
Asn-Ser-Thr and Asn-Ala-Thr (thus introducing two new
potential glycosylation sites ¯anking the H3 segment).
The modi®ed lep gene was re-cloned into a pGEM1derived vector for the in vitro expression experiments.
During this cloning step, the 50 end of the lep gene was
modi®ed such that it acquired a 50 XbaI site and ``Kozak
consensus'' sequence (Kozak, 1992), as described
(Nilsson & von Heijne, 1993).
For all L ! X and LL ! XX mutations (where X is
any of the 20 natural amino acids), Leu20 in the poly
(Leu) stretch was replaced by X or XX using the QuickChange site-directed mutagenesis kit from Stratagene.
The 6 K mutation was made by replacing the Leu19Leu22 stretch by six lysine residues.
To study turn formation in the H3 segment (including
its ¯anking regions) when it is placed in the H2 position,
the entire region between a BclI site in lep codon 59
(three codons upstream of the poly(Leu) H2 segment) in
the 1P mutant used in (Monne et al., 1999a) and a SmaI
site located downstream of the termination codon in lep
was replaced by a region from the 1P mutant constructed in this work starting 22 residues upstream of
the H3 segment and ending at the SmaI site at the 30 end
of the lep gene.
Expression in vitro
The constructs in pGEM1 were transcribed by SP6
RNA polymerase for one hour at 37 C. The transcription
mixture was as follows: 1-5 mg of DNA template, 5 ml of
10 SP6 H-buffer (400 mM Hepes-KOH (pH 7.4),
60 mM magnesium acetate, 20 mM spermidine-HCl),
5 ml of BSA (1 mg/ml), 5 ml of m7G(50 )ppp(50 )G
(10 mM), 5 ml of DTT (50 mM), 5 ml of rNTP mix
(10 mM ATP, 10 mM CTP, 10 mM UTP, 5 mM GTP),
18.5 ml of water, 1.5 ml of RNase inhibitor (50 units),
0.5 ml of SP6 RNA polymerase (20 units). Translation
was performed in reticulocyte lysate in the presence and
absence of dog pancreas microsomes (LiljestroÈm &
Garoff, 1991). Translation products were analyzed by
SDS-PAGE and gels were quantitated on a Fuji BAS1000
phosphoimager using the MacBAS 2.31 software. Glycosylation ef®ciencies were calculated as the quotient
between the intensity of the band corresponding to the
relevant glycosylated protein species divided by the
summed intensities of all glycosylated and non-glycosylated species.
196
Cytoplasmic Turns in Membrane Proteins
Figure 6. Model for helical hairpin formation with a lumenal turn
(middle), and a cytoplasmic turn
(bottom) during protein insertion
into the ER membrane (adapted
from Mothes et al., 1997). In the top
panel, there is no turn-inducing
residue in the poly(Leu) stretch
(assumed to be in the H2 position),
and only a single transmembrane
segment is formed. Formation of a
lumenal turn by a turn-inducing
residue (*) in the middle of H2 is
facilitated by the looped conformation assumed by H2 upon entering the translocation channel
(middle panel). Formation of a
cytoplasmic turn in H3 is less ef®cient, since H3 does not need to
assume a looped conformation
during membrane insertion (bottom
panel). The opening and closing of
the translocation channel during
membrane protein integration as
envisaged by Liao et al. (1997) is
indicated.
Acknowledgments
This work was supported by grants from the Swedish
Cancer Foundation and the Swedish Natural and Technical Sciences Research Councils (to G.vH.). Dog pancreas
microsomes were a kind gift from Dr M. Sakaguchi,
Fukuoka.
References
Do, H., Falcone, D., Lin, J., Andrews, D. W. & Johnson,
A. E. (1996). The cotranslational integration of
membrane proteins into the phospholipid bilayer is
a multistep process. Cell, 85, 369-378.
Engelman, D. M. & Steitz, T. A. (1981). The spontaneous
insertion of proteins into and across membranes:
the helical hairpin hypothesis. Cell, 23, 411-422.
Hamman, B. D., Hendershot, L. M. & Johnson, A. E.
(1998). BiP maintains the permeability barrier of the
ER membrane by sealing the lumenal end of the
translocon pore before and early in translocation.
Cell, 92, 747-758.
Johansson, M., Nilsson, I. & von Heijne, G. (1993). Positively charged amino acids placed next to a signal
sequence block protein translocation more ef®-
ciently in Escherichia coli than in mammalian microsomes. Mol. Gen. Genet. 239, 251-256.
Kozak, M. (1992). Regulation of translation in eukaryotic
systems. Annu. Rev. Cell Biol. 8, 197-225.
Liao, S., Lin, J., Do, H. & Johnson, A. (1997). Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome
during membrane protein integration. Cell, 90, 3141.
LiljestroÈm, P. & Garoff, H. (1991). Internally located
cleavable signal sequences direct the formation of
Semliki Forest virus membrane proteins from a
polyprotein precursor. J. Virol. 65, 147-154.
MonneÂ, M., Hermansson, M. & von Heijne, G. (1999a).
A turn propensity scale for transmembrane helices.
J. Mol. Biol. 288, 141-145.
MonneÂ, M., Nilsson, I., Elofsson, A. & von Heijne, G.
(1999b). Turns in transmembrane helices: determination of the minimal length of a ``helical hairpin''
and derivation of a ®ne-grained turn propensity
scale. J. Mol. Biol. 293, 807-814.
Mothes, W., Heinrich, S., Graf, R., Nilsson, I., von
Heijne, G., Brunner, J. & Rapoport, T. (1997). Molecular mechanisms of membrane protein integration
into the endoplasmic reticulum. Cell, 89, 523-533.
Cytoplasmic Turns in Membrane Proteins
Nilsson, I. & von Heijne, G. (1993). Determination of the
distance between the oligosaccharyltransferase
active site and the endoplasmic reticulum membrane. J. Biol. Chem. 268, 5798-5801.
Nilsson, I. & von Heijne, G. (1998). Breaking the camel's
back: proline-induced turns in a model transmembrane helix. J. Mol. Biol. 284, 1185-1189.
SaÈaÈf, A., Wallin, E. & von Heijne, G. (1998). Stop-transfer function of pseudo-random amino acid segments
during translocation across prokaryotic and eukaryotic membranes. Eur. J. Biochem. 251, 821-829.
197
von Heijne, G. & Gavel, Y. (1988). Topogenic signals in
integral membrane proteins. Eur. J. Biochem. 174,
671-678.
Wallin, E. & von Heijne, G. (1998). Genome-wide analysis of integral membrane proteins from eubacterial,
archaean, and eukaryotic organisms. Protein Sci. 7,
1029-1038.
Wolfe, P. B., Wickner, W. & Goodman, J. M. (1983).
Sequence of the leader peptidase gene of Escherichia
coli and the orientation of leader peptidase in the
bacterial envelope. J. Biol. Chem. 258, 12073-12080.
Edited by F. Cohen
(Received 16 February 2000; received in revised form 24 May 2000; accepted 30 May 2000)
III
doi:10.1006/jmbi.2001.5108 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 313, 1171–1179
Formation of Helical Hairpins during Membrane
Protein Integration into the Endoplasmic Reticulum
Membrane. Role of the N and C-terminal
Flanking Regions
Marika Hermansson, Magnus Monne and Gunnar von Heijne*
Department of Biochemistry
and Biophysics, Stockholm
University, S-106 91
Stockholm, Sweden
The helical hairpin, two closely spaced transmembrane helices separated
by a short turn, is a common structural element in integral membrane
proteins. Previous studies on the sequence determinants of helical hairpin
formation have focussed on the role of polar and charged residues placed
centrally in a long stretch of hydrophobic residues, and have yielded a
``propensity scale'' for the relative ef®ciency with which different residues promote the formation of helical hairpins. In this study, we shift
our attention to the role of charged residues ¯anking the hydrophobic
stretch. Clusters of charged residues are known to hinder membrane
translocation, and thus ¯anking charged residues may conceivably force
a long hydrophobic segment to form a helical hairpin even if there are
no or only weakly turn-promoting residues in the hydrophobic stretch.
We indeed ®nd that Lys and, more surprisingly, Asp residues strongly
affect helical hairpin formation when placed next to a poly-Leu-based
transmembrane segment. We also ®nd that a cluster of four consecutive
Lys residues can affect the ef®ciency of helical hairpin formation even
when placed 30 residues downstream of the hydrophobic stretch. These
observations have interesting implications for the way we picture
membrane protein topogenesis within the context of the endoplasmic
reticulum (ER) translocon.
# 2001 Academic Press
*Corresponding author
Keywords: membrane protein; protein structure; glycosylation;
transmembrane helix; helical hairpin
Introduction
The helical hairpin, i.e. two closely spaced
hydrophobic transmembrane helices separated by
a short turn,1 is a common structural element in
integral membrane proteins and is thought to
serve as an important ``topogenic element'' during
membrane protein assembly.2 In previous studies,
we have shown that the ef®ciency of formation of
helical hairpins during protein insertion into the
endoplasmic reticulum (ER) membrane depends
both on the overall length of the hydrophobic segment and on the identity of the central, potentially
turn-forming residues.3 ± 6 Thus, charged, polar,
and ``classical'' helix-breaking residues are good
turn-formers and effectively induce helical hairpin
formation when placed near the middle of a suf®E-mail address of the corresponding author:
[email protected]
0022-2836/01/051171±9 $35.00/0
ciently long hydrophobic stretch, whereas centrally
placed apolar residues do not induce helical hairpin formation but rather cause the hydrophobic
stretch to insert as a single, long transmembrane
helix that spans the membrane only once.
While the basic sequence determinants behind
the formation of helical hairpins are thus beginning
to be mapped out, one aspect that has so far not
been addressed is the possible role of the residues
that ¯ank the hydrophobic stretch. As charged residues are known to hinder membrane translocation
when present in certain sequence contexts, and
thus to act as powerful topological determinants in
both prokaryotic and eukaryotic membrane
proteins,2 we considered the possibility that
charged residues in the regions ¯anking a long
hydrophobic stretch may speci®cally affect helical
hairpin formation. We now report that both Lys
and Asp residues strongly affect helical hairpin formation when present close to a poly-Leu hydro# 2001 Academic Press
1172
phobic stretch, the formation of a helical hairpin
with a lumenally oriented turn is promoted by
C-terminally ¯anking charged residues, whereas
the formation of a helical hairpin with a cytoplasmically oriented turn is prevented by such ¯anking
residues. We also ®nd that a cluster of four consecutive Lys residues can affect the ef®ciency of
helical hairpin formation even when placed 30
residues downstream of the hydrophobic stretch.
These ®ndings show that a helical hairpin consists
of a long hydrophobic stretch with a central tight
turn, and a rather extended region of ¯anking residues must be included in the de®nition of this
basic ``folding unit'' in integral membrane proteins.
Results
Model proteins and topology assay
As in our previous studies of helical hairpin formation in transmembrane helices, we have used
Helical Hairpins in Membrane Proteins
the well-characterized Escherichia coli inner membrane protein leader peptidase (Lep) as a model
protein. Lep consists of two transmembrane segments (H1 and H2) connected by a short cytoplasmic loop (P1) and followed by a large
C-terminal periplasmic domain (P2). When
expressed in vitro in the presence of dog pancreas
microsomes, Lep adopts the same membrane topology as in its natural environment in the inner
membrane of E. coli,7 i.e. with the N and C termini
on the lumenal side,8 Figure 1(a) (left).
To study the formation of helical hairpins with
the short turn oriented towards the lumenal side of
the ER membrane, H2 was substituted by poly-Leu
stretches of the general composition ...P58-LI
K4L29VL10Q3P-E82... (superscript numbers refer to
residues in Lep , subscripts indicate the number of
consecutive residues of a given kind), cf. MonneÂ
et al.4,5 We have shown that this stretch forms a
single transmembrane segment when the model
protein is integrated in vitro into dog pancreas
Figure 1. Lys and Asp residues at the C-terminal end of H2 promote the formation of helical hairpins with a lumenal turn. (a) The model protein used in this study. The H2 transmembrane segment in Lep (white) was replaced with
three different poly-Leu-based segments of the composition K4L29VL10Q3P, K4L21WL7VL10Q3P, and K4L17PL7VL5Q3P,
and one to four Lys or Asp residues were introduced at the C-terminal end of the poly-Leu stretch. A glycosylation
acceptor site was placed 20 residues downstream of H2 (counting from the ®rst Gln after the hydrophobic stretch).
Depending on the lumenal or cytoplasmic localization of the P2 domain, the glycosylation acceptor site will either be
modi®ed (®lled Y) or not (open Y). (b) L17PL7VL5-derived constructs with different numbers of C-terminally ¯anking
Lys residues were translated in vitro in the absence (ÿ) and presence (‡) of rough microsomes (RM) and analyzed by
SDS-PAGE. Black and white dots indicate the glycosylated and non-glycosylated forms of the proteins, respectively.
(c) Quanti®cation of the full set of data. The percentage glycosylation was calculated as 100 I‡/(I‡ ‡ Iÿ), where
I‡ (Iÿ) is the intensity of the glycosylated (non-glycosylated) band.
Helical Hairpins in Membrane Proteins
microsomes, but that the introduction of ``turnpromoting'' residues such as Pro, Asn, Arg, or Asp
near the middle of the poly-Leu stretch leads to the
formation of a helical hairpin.5 As an easily scored
marker for the lumenal or cytoplasmic localization
of the P2 domain, an N-glycosylation site (Asn-SerThr) was introduced 20 amino acid residues downstream of H2; in constructs where the poly-Leu
stretch spans the membrane only once, this site
will be glycosylated by the lumenally disposed oligosaccharyl transferase enzyme (Figure 1(a), left),
while it will not be modi®ed in constructs where
the poly-Leu stretch has been mutated to form a
helical hairpin (Figure 1(a), right). We have shown,
using alkaline extraction, that poly-Leu-based helical hairpins are properly assembled into the ER
membrane, ruling out the possibility that low
levels of glycosylation are caused by a failure in
membrane integration rather than helical hairpin
formation.9 Turn formation can thus be assessed
by in vitro transcription/translation of the relevant
constructs in the presence of microsomes followed
by quanti®cation of the ef®ciency of glycosylation
of the engineered N-glycosylation site.
A similar strategy was used to study the formation of helical hairpins with the turn oriented
towards the cytoplasmic side of the ER membrane
(see Figure 4(a)).6 In this case, an extra transmembrane segment (H3) composed of a 40 residue
poly-Leu stretch of the composition ...E225-TSL40RSV233... was introduced into the periplasmic P2
domain. This poly-Leu stretch is long enough to
form either a single transmembrane segment or a
helical hairpin, depending on the identity of residues placed near the middle of the hydrophobic
stretch. The topology of the poly-Leu stretch can
be determined easily by analyzing the glycosylation status of two strategically placed N-glycosylation acceptor sites, one upstream and one
downstream of the hydrophobic stretch. Only the
®rst site will be modi®ed in constructs where the
poly-Leu stretch spans the membrane once
(Figure 1(a), left), whereas both will be modi®ed
when a helical hairpin is formed (Figure 4(a),
right).
C-terminally flanking Lys and Asp residues
promote the formation of helical hairpins with
a lumenally oriented turn
As a ®rst test of the effect of ¯anking charged
residues on helical hairpin formation, we studied
three constructs where the H2 transmembrane
segment in Lep was replaced by poly-Leu-based
segments of the composition K4L29VL10Q3P,
K4L21WL7VL10Q3P, and K4L17PL7VL5Q3P. As
shown previously,5 the ®rst, uniformly hydrophobic stretch forms a single transmembrane segment, while the two other constructs both have a
mixed topology with a helical hairpin formed in
approximately 50 % of the molecules. The overall
length of the hydrophobic segment (31 residues)
in the K4L17PL7VL5Q3P construct is close to the
1173
minimum length required for helical hairpin
formation.5
One to four positively charged Lys residues
were inserted immediately downstream of the
three hydrophobic stretches (replacing one to four
residues of the Q3P sequence: KQ2P, K2QP, K3P,
K4) and helical hairpin formation was assayed by
determining the fraction of glycosylated molecules,
Figure 1(b). For all three constructs, the fraction of
glycosylated molecules decreases with the number
of C-terminally ¯anking Lys residues, Figure 1(c)
(left panel). Two extra lysine residues are suf®cient
to ensure fully ef®cient helical hairpin formation in
the L17PL7VL5 construct, and a signi®cant degree
of helical hairpin formation is evident even in the
uniformly hydrophobic L29VL10 construct when
four lysine residues are added.
Since the difference in degree of helical hairpin
formation between the two ``hairpin-prone'' and
the uniformly hydrophobic segments was maximal
for three added lysine residues, we compared helical hairpin formation in the absence and presence
of three C-terminally ¯anking Lys residues for an
additional series of constructs with the general
composition K4L21XL7VL10K3P (where X is any of
the 20 natural amino acids). The X residues were
chosen on the basis of our earlier results with the
K4L21XL7VL10Q3P constructs4,5 to re¯ect the relative
ef®ciency of helical hairpin formation induced by
hydrophobic and mildly polar amino acids. As
seen in Figure 2, the effect of adding three C-terminal lysine residues is minor for the hydrophobic
residues, but is quite dramatic for Trp, Ser, Cys,
and Thr (compare the white and black bars). As a
comparison, Figure 2 also shows previously
obtained results5 for the same poly-Leu stretch
lacking ¯anking charged residues, but with a pair
of residues inserted in the middle of the stretch
(gray bars). From this comparison, it is clear that
the effect of three ¯anking lysine residues is similar
to that seen when a second polar X residue is introduced near the middle of the hydrophobic stretch.
We also tested the effects on helical hairpin formation by negatively charged Asp residues. Again,
the K4L29VL10Q3P and K4L21WL7VL10Q3P constructs were used, and the Q3P sequence was progressively replaced by one to four aspartic acid
residues, Figure 1(c) (right panel). While overall
the effects were similar to those seen for Lys (cf.
Figure 1(c), left panel), Asp appears to promote
helical hairpin formation somewhat more ef®ciently than Lys for the uniformly hydrophobic
K4L29VL10Q3P construct, while the differences seen
for the K4L21WL7VL10Q3P construct are less conspicuous.
Finally, we compared the effects of N and Cterminally ¯anking Asp and Lys residues on helical hairpin formation in a X4L29VL10Z4 construct
(X, Z ˆ K or D), where four consecutive Lys or
Asp residues were inserted in all four combinations immediately upstream and downstream of
the hydrophobic stretch, Figure 3. Quanti®cation of
the degree of glycosylation of the various con-
1174
Helical Hairpins in Membrane Proteins
Figure 2. Helical-hairpin formation in H2-segments of the general composition K4L21XL7VL10K3P
(black bars), where X is any of the
20 natural amino acids. Results
from our earlier turn propensity
study,5 where H2 was substituted
by a stretch of the design
K4L21XL7VL10Q3P (white bars) or
K4L20X2L7VL10Q3P (gray bars) are
included for comparison.
structs revealed that the identity of the N-terminally ¯anking charged residues made no difference,
ruling out an effect dependent on, e.g. charge-pairing between the N and C-terminal ¯anking
regions.
To make sure that the low levels of glycosylation seen for the these constructs were not
caused by a failure of the helical hairpin to
insert into the membrane, we expressed a truncated form of the K4L29VL10D4 construct where
residues 5-46 (including the H1 transmembrane
segment) were deleted, thus leaving the L29VL10
stretch as the only potential membrane-spanning
segment. As expected, the molecules were not
glycosylated and were associated with the membrane pellet after alkaline extraction of the
Figure 3. N-terminally ¯anking
residues do not affect helical hairpin formation in H2 (white). Four
Lys or four Asp residues were
introduced simultaneously at both
ends of an H2-stretch of the composition L29VL10, and the degree of
glycosylation was determined for
each combination of K4 and D4
¯anking segments.
1175
Helical Hairpins in Membrane Proteins
microsomes,10 thus demonstrating ef®cient membrane integration (data not shown).
We conclude that the ef®ciency of formation of
helical hairpins with a lumenally oriented turn is
strongly affected by the presence of C-terminally
(but not N-terminally) ¯anking charged residues in
our model protein, that a helical hairpin can be
induced even in a uniformly hydrophobic segment
provided that the number of C-terminally ¯anking
charged residues is suf®ciently high, and that the
ef®ciency of helical hairpin formation can be
increased signi®cantly by ¯anking charged residues in constructs such as K4L17PL7VL5Q3P, where
the overall length of the hydrophobic segment is
close to the minimum length required for the formation of a helical hairpin. Furthermore, both positively and negatively charged C-terminally
¯anking residues promote helical hairpin formation in this context, i.e. the sign of the charge
has little effect. It should be noted, though, that the
lumenal or cytoplasmic location of the N-terminal
end of the poly-Leu segment may be ®xed by the
topology of the N-terminal part of the protein
(i.e. the H1-P1 part), and thus that the effects of
N-terminally ¯anking residues may well be
different in constructs where the poly-Leu stretch
is at the N terminus.
C-terminally flanking Lys and Asp residues
inhibit the formation of helical hairpins with a
cytoplasmically oriented turn
We have shown that helical hairpins with a cytoplasmically oriented turn can be induced in a long
poly-Leu stretch by the introduction of pairs or triplets of polar or charged residues near the middle
of the hydrophobic stretch.6 To study the effect of
C-terminally ¯anking charged residues in this context, we used a construct with a poly-Leu-based
stretch of the composition TSL19P2L19RS placed in
the middle of the P2 domain, and the topology of
this stretch (a single transmembrane span or a helical hairpin) was determined by assessing the glycosylation status of two N-glycosylation acceptor
sites ¯anking the hydrophobic stretch, Figure 4(a).
The L19P2L19 segment was chosen because it is
poised near the threshold for helical hairpin formation and has an intermediate level of doubly
glycosylated molecules (56 %).
Again, from one to four Lys or Asp residues
were introduced by replacing one to four residues
immediately C-terminally to the L19 P2L19 stretch,
and the fraction of doubly versus singly glycosylated molecules was determined, Figure 4(b). As
seen in Figure 4(c), the degree of helical hairpin
formation decreased with increasing numbers of
charged residues in this case, and the effect of Lys
was somewhat stronger than that of Asp. We conclude that C-terminally ¯anking charged residues
inhibit the formation of helical hairpins with a
cytoplasmically oriented turn.
The effect on helical hairpin formation of
C-terminally flanking Lys and Asp residues
decreases with the distance from the
hydrophobic stretch
Finally, we studied the effect of increasing the
separation between the charged residues (four Lys
or four Asp) and the hydrophobic stretch for both
``lumenal turn'' (K4L29VL10Q3P construct) and
``cytoplasmic turn'' (TSL19P2L19RS construct) helical
hairpins. As shown in Figure 5, the K4 segment
promoted the formation of a helical hairpin with a
lumenally oriented turn even when placed 32 residues downstream of the hydrophobic stretch (left
panel), while the effect of a D4 segment decreased
much more rapidly with the separation distance.
Similarly, the K4 segment inhibited the formation
of a helical hairpin with a cytoplasmically oriented
turn up to larger separation distances than did the
D4 segment, although the effect drops off more
rapidly in this case (right panel). Thus, the ef®ciency of helical hairpin formation can depend on
sequence determinants located quite far away from
the hydrophobic transmembrane segment.
Discussion
The helical hairpin appears to be a basic folding
unit in multi-spanning integral membrane proteins,
and is thus central to our understanding of membrane protein topology. Based on analyses of
model membrane proteins speci®cally designed to
facilitate the study of helical hairpin formation
during membrane protein insertion into the ER
membrane, we previously quanti®ed the relative
propensities of the 20 natural amino acids to
induce the formation of helical hairpins when
placed in the middle of a long poly-Leu stretch,
both for helical hairpins with lumenally and cytoplasmically oriented turns.4 ± 6,9 We have also determined the minimal length of the hydrophobic
segment required for ef®cient helical hairpin formation.5
Membrane protein topology is, however, not
determined by only the hydrophobic transmembrane helices themselves. Charged residues in
short loops connecting the hydrophobic segments
are potent topogenic determinants; the best-known
effect in this regard is codi®ed in the so-called
positive inside rule, which states that positively
charged loops tend to remain on the cytoplasmic
side of the membrane.11,12 Based on this premise,
we hypothesized that the tendency for helical hairpin formation in a long hydrophobic stretch would
depend on the number of charged residues in the
immediate ¯anking regions, in addition to the
characteristics of the hydrophobic stretch itself.
In this study, we have thus asked whether helical hairpin formation can be in¯uenced by ¯anking
positively and negatively charged residues (Lys
and Asp). We ®nd that N-terminally ¯anking residues have no effect on helical hairpin formation in
our model protein (possibly because the lumenal
1176
Helical Hairpins in Membrane Proteins
Figure 4. Lys and Asp residues at the C-terminal end of H2 inhibit the formation of helical hairpins with a cytoplasmic turn. (a) A 40 residue poly-Leu stretch (H3, white) was inserted into the middle of the P2 domain, and the
glycosylation status of two Asn-X-Thr glycosylation acceptor sites ¯anking H3 was determined after in vitro translation in the presence of dog pancreas microsomes (glycosylated and non-glycosylated glycosylation acceptor sites
are indicated, respectively, by ®lled Y and open Y). Constructs where H3 forms a single transmembrane segment
(left) will be glycosylated on only one site, whereas those where H3 forms a helical hairpin (right) will be glycosylated on both sites. (b) TSL19P2L19RS-derived constructs with different numbers of C-terminally ¯anking Asp residues
were translated in vitro in the absence (ÿ) and presence (‡) of rough microsomes (RM) and analyzed by SDS-PAGE.
Molecules with zero, one, and two modi®ed glycosylation sites are indicated by one white dot, one black dot, and
two black dots, respectively. (c) Quanti®cation of the full set of data for C-terminally ¯anking Lys (white dots) and
Asp (black dots) residues. The percentage of doubly glycosylated molecules was calculated as the quotient between
the intensity of the doubly glycosylated band divided by the summed intensities of the doubly and singly glycosylated bands.
or cytoplasmic location of the N terminus of the
particular hydrophobic stretch studied here is
determined by the topology of the preceding part
of the model protein), whereas charged residues of
either sign placed C-terminally to the hydrophobic
stretch generally promote the cytoplasmic location
of the C-terminal end of the hydrophobic stretch.
Thus, charged C-terminally ¯anking residues promote helical hairpin formation in constructs where
the turn is oriented towards the lumenal side of
the ER membrane, and weaken the tendency to
form helical hairpins in constructs where the turn
is oriented towards the cytoplasmic side.
Interestingly, ¯anking positively and negatively
charged residues have similar effects on helical
hairpin formation when located close to the hydrophobic stretch (Figures 1 and 4), suggesting that
they somehow ``sense'' the sidedness of the ER
membrane during the co-translational membrane
assembly of the model protein. Given the high
hydrophobicity of the poly-Leu stretch used in
these studies, it is possible that the hydrophobic
stretch partitions into the lipid environment almost
immediately upon entering the protein-conducting
translocon channel in the ER membrane,13,14 thus
facilitating interactions between the charged ¯anking residues and the lipid head groups. The surprising observation that an effect on the formation
of a helical hairpin with a lumenal turn of a stretch
of four Lys residues can be detected even at quite
large separation distances (30 residues; Figure 5)
between the hydrophobic stretch and the charge
cluster further shows that a considerable length of
nascent chain can impact the process of hairpin for-
1177
Helical Hairpins in Membrane Proteins
Figure 5. The effect on helical
hairpin formation of C-terminally
¯anking Lys and Asp residues
decreases with the distance from
the hydrophobic stretch. Four Lys
(white dots) or Asp (black dots)
residues were placed at different
distances C-terminally to the H2
L29VL10 stretch (left panel) or the
H3 L19 P2L19 stretch (right panel).
The percentage singly (left panel)
or doubly (right panel) glycosylated molecules were calculated as
for Figures 1 and 4, respectively.
mation. This is somewhat reminiscent of the socalled charge block on protein translocation
observed when a string of positively charged residues is placed downstream of a signal-anchor
sequence15,16 and indicates that a fairly sizeable
portion of the nascent chain (the 40 residue hydrophobic stretch and at least 30 additional residues)
may be present within the translocon when the
helical hairpin forms. An alternative, though perhaps less likely, possibility is that a block of
charged residues located in the ribosomal tunnel
can trigger a change in the ribosome-translocon
interaction that facilitates the helical hairpin formation, cf. Liao et al.17
The stronger effect on helical hairpin formation
seen for positively charged Lys residues compared
to the negatively charged Asp residues, both for
helical hairpins with a cytoplasmic turn (Figure 4),
and for helical hairpins with a lumenal turn at
large separation distances (Figure 5), is in accord
with the positive inside rule. From this point of
view, it is surprising that the effects on helical hairpin formation exerted by negatively charged residues located close to the hydrophobic segment are
very similar or even stronger than those seen for
positively charged residues (Figure 1). In previous
studies, negatively charged residues have been
found to have only weak effects on the translocation of N-terminal tails across the membrane,18,19
and on the translocation of C-terminal domains
when placed downstream of a hydrophobic signalanchor sequence.20 We have no good explanation
for their much stronger effects on helical hairpin
formation.
In summary, our results show that ¯anking residues need to be included in the de®nition of the
helical hairpin when viewed as a fundamental
folding unit in integral membrane proteins. As a
®rst approximation, membrane proteins may thus
be pictured as being composed of single transmembrane helices (and their immediate ¯anking segments) spaced far apart in the sequence and of
helical hairpins (again including their immediate
¯anking segments). This view rests on the assumption that the topology of a (long or short) hydro-
phobic segment can be in¯uenced directly only by
the part of the nascent chain that is present within
the ribosome-translocon channel at the time when
the segment integrates into the lipid bilayer, and
thus that topology is, to an important extent, determined locally. The topological determinants for
single transmembrane helices are quite well understood,21 and our recent work on helical hairpin formation has now shed some light on this second
kind of topogenic element. Finally, we note that
the observations reported here may be relevant for
improving current methods of membrane protein
topology prediction,22 since these methods do not
distinguish between single transmembrane helices
and helical hairpins.
Materials and Methods
Enzymes and chemicals
Unless stated otherwise, all enzymes were from
Promega (Madison, WI, USA). Phage T7 DNA polymerase, BclI, [35S]Met, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, and the cap analog
m7G(50 )ppp(50 )G were from Amersham-Pharmacia
(Uppsala, Sweden). Plasmid pGEM1, transcription buffer
and rabbit reticulocyte lysate were from Promega. Oligonucleotides were from Cybergene (Stockholm, Sweden).
DNA manipulations
For cloning into and expression from the pGEM1 plasmid, the 50 end of the lep gene was modi®ed, ®rst, by the
introduction of an XbaI site and, second, by changing the
context 50 to the initiator ATG codon to a ``Kozak consensus'' sequence.23 Thus, the 50 region of the gene was
modi®ed to:
...ATAACCCTCTAGAGCCACCATGGCGAAT...
XbaI site and initiator codon underlined.
Replacement of the H2 region in Lep was performed
as described,9 i.e. by ®rst introducing BclI and NdeI
restriction sites in codons 59 and 80 ¯anking the H2
region and then replacing the BclI-NdeI fragment by the
appropriate double-stranded oligonucleotides. Sitespeci®c mutagenesis used to add BclI and NdeI restriction sites at the 30 and 50 ends of H2 in Lep and to introduce an Asn±Ser-Thr acceptor site for N-linked
glycosylation was performed according to the method of
1178
Kunkel.24,25 The glycosylation acceptor site was designed
as described,26 i.e. by replacing three codons positioned
20 codons downstream of H2 with codons for the acceptor tripeptide Asn-Ser-Thr. In all constructs, the naturally
occurring glycosylation site at Asn214 in Lep was
removed by an Asn214 ! Gln mutation. Residues 59-81
in H2 were replaced by a poly-Leu sequence of the
design LIK4L29VL10Q3P (subscripts indicate the number
of consecutive residues) for the 40 residue poly-Leu construct. In the 31 residue poly-Leu constructs, four leucine
residues were deleted from the N terminus and ®ve
leucine residues from the C terminus of the 40 residue
poly-Leu stretch by PCR mutagenesis.
Introduction of the H3 region into the periplasmic P2
domain of Lep was performed as described,6 i.e. by
using a pING1 vector containing a lep gene with SpeI
and BglII restriction sites introduced into codons 226-227
and 231-232, and then replacing the SpeI-BglII fragment
by a double-stranded oligonucleotide encoding the
sequence ...E225-TSL19P2L19RS-V233... (superscript numbers refer to the wild-type Lep sequence) ¯anked by SpeI
and BamHI (compatible with BglII) restriction sites. The
lep gene was further modi®ed by an Asn214 ! Gln
mutation (to remove a potential glycosylation site present in the wild-type Lep sequence) and by mutations
converting residues 96-98 and 258-260 to, respectively,
Asn-Ser-Thr and Asn-Ala-Thr (thus introducing two new
potential glycosylation sites ¯anking the H3 segment).
The modi®ed lep gene was re-cloned into the pGEM1derived vector described above for the in vitro expression
experiments.
The QuickChange site-directed mutagenesis kit (Stratagene) was used for the introduction of ¯anking
charged Lys and Asp residues and all L ! X substitutions. Asp residues ¯anking the H2 poly-Leu stretches
on the N-terminal side were introduced by replacing the
K4 segment by a D4 segment; Lys and Asp residues
¯anking the H2 poly-Leu stretches on the C-terminal
side were introduced by replacing the Q3P segment
(KQ2P, K2QP, K3P, K4, and the same for D); and Lys and
Asp residues ¯anking the H3 poly-Leu stretch on the Cterminal side were introduced by replacing one to four
residues immediately following the poly-Leu stretch. All
mutants were con®rmed by DNA sequencing using T7
DNA polymerase.
Expression in vitro
The constructs in pGEM1 were transcribed by SP6
RNA polymerase for one hour at 37 C. The transcription
mixture was as follows: 1-5 mg of DNA template, 5 ml of
10 SP6 H-buffer (400 mM Hepes-KOH (pH 7.4),
60 mM magnesium acetate, 20 mM spermidine-HCl),
5 ml of 1 mg/ml BSA, 5 ml of 10 mM m7G(50 )ppp(50 )G,
5 ml of 50 mM DTT, 5 ml of rNTP mix (10 mM ATP,
10 mM CTP, 10 mM UTP, 5 mM GTP), 18.5 ml of water,
1.5 ml of 33 units/ml RNase inhibitor, 0.5 ml of 40 units/
ml SP6 RNA polymerase. Translation of 1 ml of mRNA
was performed as described27 at 30 C for one hour in
9 ml of nucelase-treated reticulocyte lysate, 1 ml of 40
units/ml RNase inhibitor, 1 ml of 15 mCi/ml [35S]Met, 1 ml
of amino acids mix (1 mM of each amino acid except
Met), 1 ml of mRNA, and 1 ml of 2 units/ml dog pancreas
microsomes (one unit is de®ned as the amount of microsomes required for 50 % translocation of in vitro synthesized preprolactin). Translation products were
analyzed by SDS-PAGE and gels were quanti®ed on a
Fuji FLA-3000 phosphoimager using the Fuji Image
Helical Hairpins in Membrane Proteins
Reader 8.1j software. The glycosylation ef®ciency of a
given mutant was calculated as the quotient between the
intensity of the glycosylated band divided by the
summed intensities of the glycosylated and non-glycosylated bands for the H2-based constructs, and as the quotient between the intensity of the doubly glycosylated
band divided by the summed intensities of the doubly
and singly glycosylated bands for the H3-based constructs.
Acknowledgments
This work was supported by grants from the Swedish
Cancer Foundation and the Swedish Research Council to
G.v.H. Dog pancreas microsomes were a kind gift from
Dr M. Sakaguchi, Fukuoka.
References
1. Engelman, D. M. & Steitz, T. A. (1981). The
spontaneous insertion of proteins into and across
membranes: the helical hairpin hypothesis. Cell, 23,
411-422.
2. von Heijne, G. (2000). Recent advances in the
understanding of membrane protein assembly and
structure. Quart. Rev. Biophys. 32, 285-307.
3. Nilsson, I. & von Heijne, G. (1998). Breaking the
camel's back: proline-induced turns in a model
transmembrane helix. J. Mol. Biol. 284, 1185-1189.
4. MonneÂ, M., Hermansson, M. & von Heijne, G.
(1999). A turn propensity scale for transmembrane
helices. J. Mol. Biol. 288, 141-145.
5. MonneÂ, M., Nilsson, I., Elofsson, A. & von Heijne,
G. (1999). Turns in transmembrane helices: determination of the minimal length of a `'helical hairpin''
and derivation of a ®ne-grained turn propensity
scale. J. Mol. Biol. 293, 807-814.
6. SaÈaÈf, A., Hermansson, M. & von Heijne, G. (2000).
Formation of cytoplasmic turns between two closely
spaced transmembrane helices during membrane
protein integration into the ER membrane. J. Mol.
Biol. 301, 191-197.
7. Wolfe, P. B., Wickner, W. & Goodman, J. M. (1983).
Sequence of the leader peptidase gene of Escherichia
coli and the orientation of leader peptidase in the
bacterial envelope. J. Biol. Chem. 258, 12073-12080.
8. Nilsson, I. & von Heijne, G. (1993). Determination of
the distance between the oligosaccharyltransferase
active site and the endoplasmic reticulum membrane. J. Biol. Chem. 268, 5798-5801.
9. Nilsson, I., SaÈaÈf, A., Whitley, P., Gafvelin, G.,
Waller, C. & von Heijne, G. (1998). Proline-induced
disruption of a transmembrane a-helix in its natural
environment. J. Mol. Biol. 284, 1165-1175.
10. Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow,
P. B. (1982). Isolation of intracellular membranes by
means of sodium carbonate treatment. J. Cell. Biol.
93, 97-102.
11. von Heijne, G. & Gavel, Y. (1988). Topogenic signals
in integral membrane proteins. Eur. J. Biochem. 174,
671-678.
12. von Heijne, G. (1986). The distribution of positively
charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology.
EMBO J. 5, 3021-3027.
1179
Helical Hairpins in Membrane Proteins
13. Heinrich, S., Mothes, W., Brunner, J. & Rapoport, T.
(2000). The Sec61p complex mediates the integration
of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell, 102, 233244.
14. Mothes, W., Heinrich, S., Graf, R., Nilsson, I., von
Heijne, G., Brunner, J. & Rapoport, T. (1997). Molecular mechanisms of membrane protein integration
into the endoplasmic reticulum. Cell, 89, 523-533.
15. Andersson, H. & von Heijne, G. (1991). A 30-residue-long ``Export Initiation Domain'' adjacent to the
signal sequence is critical for protein translocation
across the inner membrane of Escherichia coli. Proc.
Natl Acad. Sci. USA, 88, 9751-9754.
16. Johansson, M., Nilsson, I. & von Heijne, G. (1993).
Positively charged amino acids placed next to a
signal sequence block protein translocation more
ef®ciently in Escherichia coli than in mammalian
microsomes. Mol. Gen. Genet. 239, 251-256.
17. Liao, S., Lin, J., Do, H. & Johnson, A. (1997). Both
lumenal and cytosolic gating of the aqueous ER
translocon pore are regulated from inside the ribosome during membrane protein integration. Cell, 90,
31-41.
18. Kiefer, D., Hu, X. T., Dalbey, R. & Kuhn, A. (1997).
Negatively charged amino acid residues play an
active role in orienting the Sec-independent Pf3 coat
protein in the Escherichia coli inner membrane.
EMBO J. 16, 2197-2204.
19. Nilsson, I. M. & von Heijne, G. (1990). Fine-tuning
the topology of a polytopic membrane protein. role
20.
21.
22.
23.
24.
25.
26.
27.
of positively and negatively charged residues. Cell,
62, 1135-1141.
Laws, J. K. & Dalbey, R. E. (1989). Positive charges
in the cytoplasmic domain of Escherichia coli leader
peptidase prevent an apolar domain from functioning as a signal. EMBO J. 8, 2095-99.
Goder, V. & Spiess, M. (2001). Topogenesis of membrane proteins: determinants and dynamics. FEBS
Letters, 504, 87-93.
MoÈller, S., Croning, M. & Apweiler, R. (2001). Evaluations of methods for the predictive evaluation of
membrane spanning regions. Bioinformatics, 17, 646653.
Kozak, M. (1992). Regulation of translation in
eukaryotic systems. Annu. Rev. Cell Biol. 8, 197-225.
Kunkel, T. A. (1987). Rapid and ef®cient site-speci®c
mutagenesis without phenotypic selection. Methods
Enzymol. 154, 367-382.
Geisselsoder, J., Witney, F. & Yuckenberg, P. (1987).
Ef®cient site-directed in vitro mutagenesis. BioTechniques, 5, 786-791.
Nilsson, I., Whitley, P. & von Heijne, G. (1994). The
C-terminal ends of internal signal and signal-anchor
sequences are positioned differently in the ER translocase. J. Cell Biol. 126, 1127-1132.
LiljestroÈm, P. & Garoff, H. (1991). Internally located
cleavable signal sequences direct the formation of
Semliki Forest virus membrane proteins from a
polyprotein precursor. J. Virol. 65, 147-154.
Edited by F. Cohen
(Received 4 June 2001; received in revised form 24 September 2001; accepted 24 September 2001)
IV
doi:10.1016/j.jmb.2003.10.019
J. Mol. Biol. (2003) 334, 803–809
Inter-helical Hydrogen Bond Formation During
Membrane Protein Integration into the ER Membrane
Marika Hermansson and Gunnar von Heijne*
Department of Biochemistry
and Biophysics, Arrhenius
Laboratory, Stockholm
University, SE-106 91
Stockholm, Sweden
Recent work has shown that efficient di- or trimerization of hydrophobic
transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as
Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane
helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen
bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a
non-aqueous subcompartment within the translocon.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: membrane protein; protein structure; hydrogen bond;
transmembrane helix; helical hairpin
Introduction
In eukaryotic cells, most integral membrane proteins insert into and fold in the membrane of the
endoplasmic reticulum (ER) from which they may
then be transported further along the secretory
pathway or to the nuclear envelope. The initial recognition of the hydrophobic segments that form
the transmembrane a-helices takes place during
polypeptide transfer through the Sec61 translocon
complex in the ER membrane.1,2 It appears that
the transmembrane helices can either be recognized and transferred laterally into the surrounding lipid bilayer one by one, or in pairs,3 – 6 or even
as higher-order multimers,7 though these early
steps on the folding pathway are only poorly
understood at present.
Recent biochemical studies of model peptides in
detergent micelles or liposomes have shed some
light on the basic physical chemistry of helix –
helix interactions in a non-polar environment.
Tight close-packing between helices mediated by
the so-called GxxxG-motif, or inter-helical hydrogen bond formation between pairs of polar residues such as Asn and Asp, or more complex
Abbreviations used: ER, endoplasmic reticulum; Lep,
leader peptidase; RM, rough microsomes.
E-mail address of the corresponding author:
[email protected]
motifs involving Thr or Ser8 – 16 have been shown
to drive efficient dimer formation in these systems.
These interactions have been confirmed by twohybrid assays in Escherichia coli where the formation of a dimeric protein complex in the inner
membrane ultimately results in the activation of a
reporter gene.17,18 It is thus quite clear that hydrogen bonding can drive helix – helix interactions in
biological membranes; what is not clear, however,
is if inter-helix hydrogen bonds can form already
within the context of the translocon.
Inspired by previous observations that suggest
that transmembrane helices may encounter a
bilayer-like environment almost immediately
upon entering the translocon channel,2,19 we have
carried out a detailed analysis of the possible influence of Asn-Asn and Asp-Asp pairs on the formation of so-called helical hairpins (i.e. two
closely spaced transmembrane helices connected
by a short loop) during co-translational membrane
protein insertion into the ER. We have already
reported that helical hairpin formation is facilitated
by the presence of charged, polar, and weakly
hydrophobic residues in the short connecting
loop,20 – 22 and by charged residues placed immediately downstream of a long, uniformly hydrophobic segment.23 Here, we show that Asn-Asn or
Asp-Asp pairs can promote the formation of helical hairpins in a strictly position-specific manner.
The observed pattern of hairpin-promoting pairs
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
804
is consistent with the formation of inter-helical
hydrogen bonds. If this interpretation is correct, it
provides yet another argument for the contention
that transmembrane helices are formed within
a non-aqueous subcompartment in the Sec61
translocon.
Results
Model protein and topology assay
As in our previous studies of helical hairpin formation in membrane proteins, we have used the
well-characterized E. coli inner membrane protein
leader peptidase (Lep) as a model protein. Lep consists of two transmembrane segments (H1 and H2)
connected by a short cytoplasmic loop (P1) and a
large C-terminal periplasmic domain (P2). When
expressed in vitro in the presence of dog pancreas
rough microsomes (RM), Lep adopts the same
membrane topology as in its natural environment
in the inner membrane of E. coli,24 i.e. with the N
terminus and the large C-terminal P2 domain on
the lumenal side,25 Figure 1(a) (left).
For the studies reported here, H2 was substituted by a poly-Leu stretch of the general composition …P58-LIK4L29VL10Q3P-E82… (superscript
numerals refer to residues in Lep, subscript
numerals indicate the number of consecutive residues of a given kind).20,22 The segment has four Nterminal lysine residues intended to anchor this
end to the cytoplasmic side of the membrane, a 40
residue stretch composed of 39 leucine and one
valine (the latter resulting from an engineered
Hydrogen Bonds in Membrane Proteins
restriction site) that is long enough to span the
membrane either as a single or as two closely
spaced transmembrane helices, and four
uncharged polar residues (Q3P) demarcating the
C-terminal end of the hydrophobic stretch. We
have shown previously that this stretch forms a
single transmembrane segment when the model
protein is integrated in vitro into RMs,26 but that
the introduction of “turn-promoting” residues
such as Pro, Asn, Arg, or Asp near the middle of
the poly-Leu stretch leads to the formation of a
helical hairpin.20
As an easily scored marker for the lumenal or
cytoplasmic localization of the P2 domain, an Nglycosylation site (Asn-Ser-Thr) was introduced 20
amino acid residues downstream of the poly-Leu
stretch. In constructs where the poly-Leu stretch
spans the membrane only once, this site will be
glycosylated by the lumenally disposed oligosaccharyl transferase enzyme (Figure 1(a), left),
while it will not be modified in constructs where
the poly-Leu stretch has been mutated to form
a helical hairpin (Figure 1(b), right).27 Turn formation can thus be assayed by in vitro transcription/translation of the relevant constructs in the
presence of RM followed by quantification of the
efficiency of glycosylation of the engineered N-glycosylation site.
Typical gels for three constructs described below
(D8, D33, and D8-D33) are shown in Figure 1(b); in
these constructs, the poly-Leu segment in D8-D33
inserts almost exclusively as a helical hairpin
(very little glycosylation), while essentially no
helical hairpin formation is seen for D8 (almost
complete glycosylation). The D33 construct has a
Figure 1. (a) The Lep model protein. Wild-type Lep has two transmembrane segments (H1, H2), and inserts into
dog pancreas rough microsomes in vitro with the Nlum –Clum orientation shown on the left. In the studies reported
here, the H2 segment has been replaced by a poly-Leu-based sequence (LIK4L29VL10Q3P) into which one or two Asn
or Asp residues have been inserted. Constructs where H2 spans the membrane once are glycosylated by the lumenally
disposed oligosaccharyl transferase enzyme on a unique Asn-Ser-Thr glycosylation acceptor site (Y) in the P2 domain
(left); those where the H2 segment forms a helical hairpin are not (right). In this study, one or a pair of Asn or Asp residues has been inserted in the areas in the H2 segment encircled on the right-hand model. (b) The D8, D33, and D8-D33
constructs were translated in vitro in the absence (2 ) and in the presence (þ) of dog pancreas rough microsomes (RM).
Unglycosylated and glycosylated molecules are indicated by the white dot and the black dot, respectively.
805
Hydrogen Bonds in Membrane Proteins
mixed topology with about half the molecules
being glycosylated.
Effects on helical hairpin formation of single
Asn and Asp residues in a (39L 1 V)
transmembrane segment
To test the effect on helical hairpin formation
when an Asn or Asp residue is placed in different
positions in a poly-Leu stretch, we introduced Asn
or Asp in positions 8, 10, 12 and 31 – 39 in the
(39L þ V) segment, and helical hairpin formation
was assayed by determining the fraction of nonglycosylated molecules ( f ng) for each construct.
None of the single Asn-mutations induce efficient formation of a helical hairpin (Figure 2(a)).
We always observe a background level of
f ng , 0.2, most likely a result of somewhat inefficient targeting and glycosylation in the in vitro system. The only positions in which an Asn leads to a
slight increase in f ng are 12 and 37. We have already
shown that an Asn-residue placed in position 22
near the middle of the (39L þ V) segment induces
almost complete formation of a helical hairpin
( f ng ¼ 0.85),22 and it is therefore not very surprising
that we see a slight effect with the N12 mutation. It
is less obvious why the N37 mutation should have
a comparable effect (but see Discussion).
For constructs with a single Asp in the (39L þ V)
stretch, the picture is the same as for Asn for
positions 8, 10, and 12 (Figure 2(b)). The Asp
mutations near the C-terminal end of the segment
have significantly higher f ng values than the corresponding Asn mutations, with the D37 mutation
having the strongest effect. It thus appears that an
Asp residue near the C-terminal end of the
(39L þ V) stretch induces a significant level of helical hairpin formation, in agreement with earlier
studies where Asp residues immediately flanking
the C-terminal end of the (39L þ V) stretch were
shown to induce a helical hairpin conformation.23
We have shown that a single Pro residue placed
10– 12 residues from either end of the (39L þ V)
stretch induces a significant amount of the helical
hairpin conformation,20 suggesting that the higher
f ng values seen for the more centrally placed D12,
D32, D33, and (possibly) D34 mutations might be
caused by the strong hairpin-inducing effect
demonstrated earlier for a centrally placed Asp
residue.20
Position-specific effects on helical hairpin
formation by Asn-Asn and Asp-Asp pairs
Figure 2. Degree of helical hairpin formation
measured as the fraction of non-glycosylated molecules
( f ng) observed after in vitro translation in the presence of
RM for single Asn (a) and single Asp (b) mutations in
the (39L þ V) H2 segment. The position of the Asn or
Asp residue is counted from the N-terminal end of the
(39L þ V) segment. All measurements are mean values
of two or three independent experiments; in general,
repeat f ng-value measurements differ by no more than
^0.05 from the mean value.
We next examined the tendencies of Asn-Asn
and Asp-Asp pairs to mediate helical hairpin formation by introducing such pairs in the (39L þ V)
stretch using the single-residue constructs reported
above for comparison.
After an initial screening of a small number of
Asn-Asn pairs, we focused mainly on the N8 position, and made a complete set of pairs with the
second Asn in positions 32 – 39. For the N10 and
N12 pairs, we made constructs with the second
Asn in positions 33, 35, 37, 38 (for N12), and 39.
Strikingly, there is an almost twofold increase in
f ng for the N8 –N33, N8 –N37, and N12 –37 constructs over the corresponding single-Asn constructs, while all the other Asn-Asn constructs
have f ng values similar to those of the corresponding single-Asn constructs.
The same set of constructs but with Asp instead
of Asn were tested (Figure 3(b)). The D8-D33 and
D8-D37 constructs have , 1.5-fold higher f ng values
than seen for the corresponding individual
mutations. In addition, the D10 – D35, D10 – D37,
and D10 –D39 constructs, and all constructs that
include D12, have higher f ng values than those of
the corresponding single-Asp constructs.
As a control to ensure that the poly-Leu segments in even the poorly glycosylated constructs
were integrated properly into the microsomal
membrane, we made a final series of constructs
where the H1 transmembrane segment and a part
806
Figure 3. Degree of helical hairpin formation
measured as the fraction of non-glycosylated molecules
( f ng) observed after in vitro translation in the presence
of RM for Asn-Asn (a) and Asp-Asp (b) pairs in the
(39L þ V) H2 segment. The positions of the Asn or Asp
residues are counted from the N-terminal end of the
(39L þ V) segment.
of P1 (residues 5 – 46) were deleted, leaving the
poly-Leu stretch as the only hydrophobic segment
in the protein. These constructs were expressed in
the presence of RM, and their membrane integration was tested by alkaline extraction of the
membranes.28,29 Two typical examples are shown
in Figure 4. The D33(DH1) construct is partially
glycosylated when expressed in the presence of
RM, and both the glycosylated and the non-glycosylated molecules remain in the membrane pellet
(lanes 3 and 4). Likewise, even though the D8D33(DH1) construct is glycosylated only weakly,
the bulk of the molecules remain in the membrane
pellet when the protein is expressed in the presence of RM. Similar results were obtained for the
other DH1 constructs tested (D33(DH1), D37(DH1),
D8-D35(DH1), and D8-D37(DH1); data not shown).
Discussion
In earlier work on helical hairpin formation, we
have shown that single polar or charged residues
Hydrogen Bonds in Membrane Proteins
introduced in the middle of a sufficiently long
poly-Leu transmembrane segment can induce an
almost complete conformational transition from a
long single-spanning transmembrane segment to a
helical hairpin.20 – 22,26,30 We have further shown
that charged residues placed immediately downstream of the poly-Leu transmembrane segment
can induce the formation of helical hairpins even
when no polar or charged residue is present within
the hydrophobic stretch itself.23
Inspired by the observation that hydrogen bonding between Asn or Asp residues in transmembrane helices in detergent micelles, model
membranes, or the E. coli inner membrane can
drive oligomerization,12,13,31 we have now tested
whether pairs of Asn or Asp residues, i.e. residues
shown to mediate strong dimerization in these
studies, can promote the formation of intramolecular helical hairpins during membrane protein
assembly into the ER membrane, i.e. within the
context of the Sec61 translocon.
As shown in Figures 2 and 3, Asn-Asn and AspAsp pairs placed in a model (39L þ V) transmembrane segment can increase the efficiency of helical
hairpin formation by 1.5– 2-fold over that seen for
the corresponding single Asn or Asp residues.
Even more strikingly, the effect is highly positionspecific. For both Asn and Asp, pairs in positions
8 –33 and 8 –37 in the 40 residue long hydrophobic
segment induce the formation of a helical hairpin,
as does the N12 – N37 pair and all pairs with Asp
in positions 10 or 12 combined with a second Asp
in positions 33, 35, 37 or 39.
The Asn and Asp 8 – 33 and 8 –37 pairs are
especially interesting, as they are clearly positionspecific (a shift of either of the two residues by
one or two positions abolishes the effect) and
further involve residues that are both one to two
turns from the ends of the (39L þ V) stretch. Moreover, if one assumes a helical hairpin conformation
for the (39L þ V) stretch, residues 33 and 37 will be
located one turn apart on the same face of the Cterminal helix of the hairpin. These pairs of residues would thus be in a perfect position to stabilize the helical hairpin conformation by interresidue hydrogen bonding.
For Asn, the only other pair that has an
increased level of helical hairpin formation compared to the two corresponding single-Asn constructs is N12 – N37. Presumably, N12 is near the
middle of the first helix in the hairpin, while N37
is close to the C-terminal end of the second helix,
making a direct interaction a less likely explanation
in this case. Since both N12 and N37 have a slightly
higher level of helical hairpin conformation than
the other single-Asn constructs, there may be an
additive effect of the two Asn residues that reflects,
e.g. interactions with the translocon rather than
inter-residue hydrogen bonding. Further studies
will be necessary to resolve this point.
The Asp-Asp pairs are less informative, since the
single-Asp mutations, in general, have a stronger
effect than the single-Asn mutations (Figure 2(b)).
807
Hydrogen Bonds in Membrane Proteins
Figure 4. Alkaline extraction of the D33(DH1) and D8-D33(DH1) constructs expressed in vitro in the absence (2) and
presence (þ ) of RM. Unglycosylated and glycosylated molecules are indicated by the white dot and the black dot,
respectively. S, supernatant; P, membrane pellet.
It is thus difficult to discern an obvious pattern in
the Asp-Asp pair constructs (Figure 3(b)). Nevertheless, it is interesting to note that helical hairpin
formation is clearly increased in the 8– 33 and 8 –
37 pairs also for Asp.
If our interpretation that the 8 –33 and 8 –37 Asn
and Asp pairs indeed stabilize the helical hairpin
conformation by hydrogen bonding is correct, it
implies, first, that the formation of the helical hairpin must take place in a non-aqueous environment
within the translocon, since stable inter-residue
hydrogen bonding would not be expected in an
aqueous environment; previous results from our
laboratory also point in this direction.21 Second, it
further implies that the two halves of the helical
hairpin formed in the (39L þ V) segment cannot
rotate freely relative to one another, as only positions on one face of the two helices (8 – 33 and 8 –
37 but not 8– 35 or 10 –33) are implicated. It is not
immediately obvious why these faces are special;
perhaps the polar residues flanking the hydrophobic stretch help orient the helices relative to
the translocon. In any case, our results suggest
that very specific helix –helix interactions can be
formed within the context of the ER translocon
and that such interactions can have a dramatic
effect on membrane protein topology.
Materials and Methods
Enzymes and chemicals
Unless stated otherwise, all enzymes were from Promega (Madison, WI, USA). [35S]Met, ribonucleotides,
deoxyribonucleotides, dideoxyribonucleotides, and the
cap analog m7G(50 )ppp(50 )G were from Amersham-Pharmacia (Uppsala, Sweden). Plasmid pGEM1, DTT, transcription buffer and rabbit reticulocyte lysate were from
Promega. Spermidine was from Sigma. Oligonucleotides
were from Cybergene (Stockholm, Sweden).
DNA manipulations
For cloning into and expression from the pGEM1 plasmid, the 50 end of the lep gene was modified, first, by the
introduction of an Xba I site and, second, by changing the
context 50 to the initiator ATG codon to a “Kozak consensus” sequence.32 Thus, the 50 region of the gene was
modified to:
…ATAACCCTCTAGAGCCACCATGGCGAAT…
(Xba I site and initiator codon underlined).
Replacement of the H2 region in Lep was performed
as described,27 i.e. by first introducing Bcl I and Nde I
restriction sites in codons 59 and 80 flanking the H2
region and then replacing the Bcl I-Nde I fragment by the
appropriate double-stranded oligonucleotides. Sitespecific mutagenesis used to add Bcl I and Nde I restriction sites at the 30 and 50 ends of H2 in Lep and to introduce an Asn-Ser-Thr acceptor site for N-linked
glycosylation was performed according to the method
of Kunkel.33,34 The glycosylation acceptor site was
designed as described,35 i.e. by replacing three codons
positioned 20 codons downstream of H2 with codons
for the acceptor tripeptide Asn-Ser-Thr. In all constructs,
the naturally occurring glycosylation site at Asn214 in
Lep was removed by an Asn214 ! Gln mutation. Residues 59 – 81 in H2 were replaced by a (39L þ V) sequence
of the design LIK4L29VL10Q3P (subscripts indicate the
number of consecutive residues).
The QuickChange site-directed mutagenesis kit (Stratagene) was used for the introduction of Asn or Asp residues in position 8, 10, and 12 and in positions 33 – 39 of
the (39L þ V) stretch. All mutants were confirmed by
DNA sequencing of plasmids using ABI PRISMw
BigDyee Terminator Cycle Sequencing Ready Reaction
Kit (Applied Biosystems).
Expression in vitro
The constructs in pGEM1 were transcribed by SP6
RNA polymerase for one hour at 37 8C. The transcription
mixture was as follows: 1– 5 mg of DNA template, 5 ml of
10 £ SP6 H-buffer (400 mM Hepes– KOH (pH 7.4),
60 mM magnesium acetate, 20 mM spermidine– HCl),
5 ml of BSA (1 mg/ml), 5 ml of 10 mM m7G(50 )ppp(50 )G,
808
5 ml of 50 mM DTT, 5 ml of rNTP mix (10 mM ATP,
10 mM CTP, 10 mM UTP, 5 mM GTP), 18.5 ml of water,
1.5 ml of RNase inhibitor (33 units/ml), 0.5 ml of SP6
RNA polymerase (40 units/ml). Translation of 1 ml of
mRNA was performed as described36 at 30 8C for one
hour in 9 ml of nuclease-treated reticulocyte lysate, 1 ml
of RNase inhibitor (40 units/ml), 1 ml of [35S]Met
(15 mCi/ml), 1 ml of amino acids mix (1 mM each amino
acid except Met), 1 ml of mRNA, and 1 ml of dog pancreas microsomes (2 units/ml; one unit is defined as the
amount of microsomes required for 50% translocation of
in vitro synthesized preprolactin). Translation products
were analyzed by SDS-PAGE and gel bands were quantified on a Fuji FLA-3000 phosphoimager using the Fuji
Image Reader 8.1j software. The glycosylation efficiency
of a given mutant was calculated as the intensity of the
glycosylated band divided by the summed intensities of
the glycosylated and non-glycosylated bands. Sodium
carbonate extraction of microsomes was carried out as
described.29
Hydrogen Bonds in Membrane Proteins
10.
11.
12.
13.
14.
Acknowledgements
This work was supported by grants from the
Swedish Cancer Foundation, the Swedish Research
Council, and the Margareta and Marcus Wallenberg Foundation to G.v.H. Dog pancreas microsomes were a kind gift from Dr M. Sakaguchi,
Fukuoka.
References
1. Goder, V. & Spiess, M. (2001). Topogenesis of membrane proteins: determinants and dynamics. FEBS
Letters, 504, 87 – 93.
2. von Heijne, G. (2003). Membrane protein assembly in
vivo. Advan. Protein Chem. 63, 1 – 18.
3. Oliver, J., Jungnickel, B., Görlich, D., Rapoport, T. &
High, S. (1995). The Sec61 complex is essential for
the insertion of proteins into the membrane of the
endoplasmic reticulum. FEBS Letters, 362, 126– 130.
4. Do, H., Falcone, D., Lin, J., Andrews, D. W. & Johnson, A. E. (1996). The cotransational integration of
membrane proteins into the phospholipid bilayer is
a multistep process. Cell, 85, 369– 378.
5. Heinrich, S., Mothes, W., Brunner, J. & Rapoport, T.
(2000). The Sec61p complex mediates the integration
of a membrane protein by allowing lipid partitioning
of the transmembrane domain. Cell, 102, 233– 244.
6. Kanki, T., Young, M. T., Sakaguchi, M., Hamasaki, N.
& Tanner, M. J. A. (2003). The N-terminal region of
the transmembrane domain of human erythrocyte
band 3—residues critical for membrane insertion
and transport activity. J. Biol. Chem. 278, 5564– 5573.
7. Borel, A. C. & Simon, S. M. (1996). Biogenesis of
polytopic membrane proteins: membrane segments
assemble with translocation channels prior to membrane integration. Cell, 85, 379– 389.
8. Russ, W. P. & Engelman, D. M. (2000). The GxxxG
motif: a framework for transmembrane helix– helix
association. J. Mol. Biol. 296, 911 – 919.
9. Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. &
Engelman, D. M. (2000). Interhelical hydrogen bond-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
ing drives strong interactions in membrane proteins.
Nature Struct. Biol. 7, 154– 160.
Senes, A., Gerstein, M. & Engelman, D. M. (2000).
Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched
residues at neighboring positions. J. Mol. Biol. 296,
921– 936.
Zhou, F. X., Merianos, H. J., Brunger, A. T. & Engelman, D. M. (2001). Polar residues drive association
of polyleucine transmembrane helices. Proc. Natl
Acad. Sci. USA, 98, 2250– 2255.
Choma, C., Gratkowski, H., Lear, J. D. & DeGrado,
W. F. (2000). Asparagine-mediated self-association of
a model transmembrane helix. Nature Struct. Biol. 7,
161– 166.
Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2001).
Polar side chains drive the association of model
transmembrane peptides. Proc. Natl Acad. Sci. USA,
98, 880– 885.
Li, R., Mitra, N., Gratkowski, H., Vilaire, G., Litvinov,
R., Nagasami, C. et al. (2003). Activation of integrin
aIIbb3 by modulation of transmembrane helix
associations. Science, 300, 795– 798.
Dawson, J. P., Melnyk, R. A., Deber, C. M. & Engelman, D. M. (2003). Sequence context strongly modulates association of polar residues in transmembrane
helices. J. Mol. Biol. 331, 255– 262.
Dawson, J., Weinger, J. & Engelman, D. (2002). Motifs
of serine and threonine can drive association of
transmembrane helices. J. Mol. Biol. 316, 799– 805.
Langosch, D., Brosig, B., Kolmar, H. & Fritz, H. J.
(1996). Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR
transcription activator. J. Mol. Biol. 263, 525–530.
Russ, W. P. & Engelman, D. M. (1999). TOXCAT: a
measure of transmembrane helix association in a biological membrane. Proc. Natl Acad. Sci. USA, 96,
863– 868.
Mothes, W., Heinrich, S., Graf, R., Nilsson, I., von
Heijne, G., Brunner, J. & Rapoport, T. (1997). Molecular mechanisms of membrane protein integration
into the endoplasmic reticulum. Cell, 89, 523–533.
Monné, M., Nilsson, I., Elofsson, A. & von Heijne, G.
(1999). Turns in transmembrane helices: determination of the minimal length of a “helical hairpin”
and derivation of a fine-grained turn propensity
scale. J. Mol. Biol. 293, 807– 814.
Nilsson, I., Johnson, A. E. & von Heijne, G. (2003).
How hydrophobic is alanine? J. Biol. Chem. 278,
29389– 29393.
Monné, M., Hermansson, M. & von Heijne, G. (1999).
A turn propensity scale for transmembrane helices.
J. Mol. Biol. 288, 141– 145.
Hermansson, M., Monné, M. & von Heijne, G. (2001).
Formation of helical hairpins during membrane protein integration into the endoplasmic reticulum
membrane. Role of the N and C-terminal flanking
regions. J. Mol. Biol. 313, 1171– 1179.
Wolfe, P. B., Wickner, W. & Goodman, J. M. (1983).
Sequence of the leader peptidase gene of Escherichia
coli and the orientation of leader peptidase in the
bacterial envelope. J. Biol. Chem. 258, 12073– 12080.
Nilsson, I. & von Heijne, G. (1993). Determination of
the distance between the oligosaccharyltransferase
active site and the endoplasmic reticulum membrane. J. Biol. Chem. 268, 5798– 5801.
Nilsson, I. & von Heijne, G. (1998). Breaking the
809
Hydrogen Bonds in Membrane Proteins
27.
28.
29.
30.
31.
camel’s back: proline-induced turns in a model
transmembrane helix. J. Mol. Biol. 284, 1185–1189.
Nilsson, I., Sääf, A., Whitley, P., Gafvelin, G., Waller,
C. & von Heijne, G. (1998). Proline-induced
disruption of a transmembrane a-helix in its natural
environment. J. Mol. Biol. 284, 1165– 1175.
Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B.
(1982). Isolation of intracellular membranes by
means of sodium carbonate treatment. J. Cell Biol.
93, 97 –102.
Sakaguchi, M., Mihara, K. & Sato, R. (1987). A short
amino-terminal segment of microsomal cytochrome
P-450 functions both as an insertion signal and as a
stop-transfer sequence. EMBO J. 6, 2425– 2431.
Sääf, A., Hermansson, M. & von Heijne, G. (2000).
Formation of cytoplasmic turns between two closely
spaced transmembrane helices during membrane
protein integration into the ER membrane. J. Mol.
Biol. 301, 191– 197.
Howard, K. P., Lear, J. D. & DeGrado, W. F. (2002).
32.
33.
34.
35.
36.
Sequence determinants of the energetics of folding
of a transmembrane four-helix-bundle protein. Proc.
Natl Acad. Sci. USA, 99, 8568– 8572.
Kozak, M. (1992). Regulation of translation in
eukaryotic systems. Annu. Rev. Cell Biol. 8, 197– 225.
Kunkel, T. A. (1987). Rapid and efficient site-specific
mutagenesis without phenotypic selection. Methods
Enzymol. 154, 367– 382.
Geisselsoder, J., Witney, F. & Yuckenberg, P. (1987).
Efficient site-directed in vitro mutagenesis. BioTechniques, 5, 786– 791.
Nilsson, I., Whitley, P. & von Heijne, G. (1994). The
C-terminal ends of internal signal and signal-anchor
sequences are positioned differently in the ER translocase. J. Cell Biol. 126, 1127– 1132.
Liljeström, P. & Garoff, H. (1991). Internally located
cleavable signal sequences direct the formation of
Semliki Forest virus membrane proteins from a polyprotein precursor. J. Virol. 65, 147– 154.
Edited by F. E. Cohen
(Received 18 June 2003; accepted 8 October 2003)
V
Confronting fusion protein-based membrane protein
topology mapping with reality: the Escherichia coli ClC
chloride channel
Marika Cassel and Gunnar von Heijne
Department of Biochemistry and Biophysics
Stockholm University
SE-106 91 Stockholm, Sweden
Corresponding author: GvH
Phone: Int+46-8-16 25 90
Fax: Int+46-8-15 36 79
E-mail: [email protected]
Keywords: membrane protein, ion channel, YadQ, topology, protein structure
Abstract
The topology of bacterial inner membrane proteins is commonly determined using
topology reporters such as PhoA and GFP fused to a series of C-terminally truncated
versions of the protein in question. Here, we report a detailed topology mapping of the
Escherichia coli inner membrane chloride channel YadQ. Since the 3D structure of
YadQ is known, our results provide a critical test of the reporter fusion approach and
offer new insights into the YadQ folding pathway.
-2-
Introduction
With the number of high-resolution membrane protein structures still hovering
around 100 and increasing only slowly, topology modeling will remain an important
aspect of membrane protein biochemistry and bioinformatics for years to come.
Theoretical topology prediction methods are being continually improved but are still far
from perfect (Chen et al. 2002). On the experimental side, reporter fusion techniques are
by far the most widely used for determining the topology of bacterial inner membrane
proteins (Manoil 1991), and can also be used in yeast (Kim et al. 2003).
Obviously, once a high-resolution structure becomes available reporter fusions
would seem to be of little value, and we do not know of a single case where an
experimental topology analysis has been done post facto. Here, we report the results of a
detailed, reporter fusion-based topology mapping of the E. coli ClC chloride channel, a
protein for which an X-ray structure was reported in 2002 (Dutzler et al. 2002). Using the
structure as a guide, we have made a set of optimally placed reporter fusions, giving us
the possibility to confront a best-case reporter fusion scenario with the hard facts of a
high-resolution structure. We find that the reporter fusion data accurately reflects the
topology of 10 of the 14 transmembrane helices, but misses two short helical hairpins on
the periphery of the protein. Given the rather weak hydrophobicity of these hairpins and
their peripheral locations, we suggest that the reporter fusion results reflect not only the
final topology of the protein but also pin-point parts of the molecule that may insert into
the membrane at a late stage in the folding process.
-3-
Results
The ClC chloride channel
The amino acid sequence, topology, and 3D structure of the E. coli ClC chloride
channel YadQ are shown in Fig. 1. The protein is a homodimer, with an ion pore in each
of the monomers. There are 14 transmembrane helices in the monomer, some of which
are very long, some very short, and some highly inclined relative to the membrane
normal. Given this rather complicated structure of the transmembrane helix bundle, we
reasoned that YadQ would be particularly challenging for topology mapping techniques
and that the results of a topology analysis may help to elucidate the propensity of the
different transmembrane helices to insert into the membrane.
Topology reporter analysis
The most widely used technique for topology mapping of bacterial inner
membrane proteins is based on reporter protein fusions to C-terminally truncated
versions of the inner membrane protein in question. We have used two well-established
topology reporter proteins, green fluorescent protein (GFP) and alkaline phosphatase
(PhoA), to map the topology of YadQ. GFP folds and becomes fluorescent only when
localized in the cytoplasm, not when located in the periplasm (Feilmeier et al. 2000;
Drew et al. 2002). PhoA behaves in the opposite way, and is enzymatically active when
located in the periplasm but not when in the cytoplasm (Manoil and Beckwith 1986).
A useful rule of thumb when making topology reporter fusions is to place the
fusion joints near the C-terminal ends of the loops between the transmembrane helices
(Boyd et al. 1993). With the YadC structure in hand, we could choose optimal fusion
joint as shown in Fig. 1A. Every loop in the structure was targeted; the final list of
fusions that were constructed is given in Table 1. GFP and PhoA activities were
measured as described previously (Rapp et al. 2004), and are also given in Table 1. In all
-4-
cases, there is a good correlation between the activities of the two reporters: fusions with
a high GFP activity invariably have a low PhoA activity, and vice versa.
Reporter fusions vs. 3D structure
The reporter fusion results are mapped onto the 3D structure in Fig. 2A and on
the corresponding topology map in Fig. 1B. In general, the activities of the GFP and
PhoA fusions correspond well with the 3D structure, but there are two obvious
exceptions where the fusion data is not in accord with the 3D structure: the E/F and P/Q
loops (the E-F and P-Q helical hairpins are highlighted in the Figure). Although these
loops are oriented towards the periplasm in the 3D structure, the GFP and PhoA fusions
consistently report cytoplasmic localizations.
From a topology mapping point of view, the best current approach is to constrain
theoretical prediction methods such as TMHMM or HMMTOP by experimental data of
the kind generated here (Melén et al. 2003). Using the full set of reporter fusion results as
constraints for TMHMM, we obtain the topology model shown in Fig. 2B (top).
Compared to the unconstrained TMHMM prediction, Fig. 2B (middle), the ends of the
transmembrane helices are much better defined in the constrained model. The
unconstrained model misses helix F, fuses helices G-H as well as O-P into single helices,
and incorrectly predicts the C-terminus to be periplasmic. The fully constrained
prediction, on the other hand, misses the E-F and P-Q hairpins.
In a more realistic setting where the 3D structure of the target protein is not
known, one generally resorts to hydrophobicity plots to guide the selection of reporter
fusion sites. Such a plot is shown in Fig. 2C, with the reporter fusions analyzed above
indicated by filled and unfilled circles. A conservative guess as to which fusions sites
would have been chosen based only on the hydrophobicity plot is shown by the arrows.
Using only the latter as constraints for TMHMM results in the topology model shown in
-5-
Fig. 2B (bottom). In this model, both the E-F and G-H hairpins are missed, the O and P
helices are merged, and the Q helix is missed.
Discussion
Among the known helix bundle membrane protein 3D structures, the chloride
channel YadQ stands out in terms of length variation among the transmembrane helices
and modes of helix-helix packing interactions (Dutzler et al. 2002). It would thus appear
to be a particularly challenging protein for topology mapping by reporter fusions, and
might pin-point particular kinds of shortcomings of this widely used approach. Seen from
a different perspective, the behavior of topology reporters fused to internal loops in the
protein might also reveal differences in membrane insertion ability between different
transmembrane helices and thereby help illuminate steps in the membrane insertion
pathway.
For these reasons, we have undertaken a detailed topology mapping of YadQ,
using complementary GFP and PhoA reporter fusions. The fusions sites were chosen
based on the 3D structure: all loops between helices (both transmembrane helices and
surface helices) were targeted, and the fusion sites were placed at the C-terminal end of
each loop, Table 1.
The results of the reporter fusion analysis are mapped onto the 3D structure in
Fig. 2A, and are also summarized in the topology model presented in Fig. 1B. Despite
the unruly 3D structure, all reporter fusions but two report the correct location of the
corresponding loop, attesting to the general reliability of the reporter fusion technique.
The fusions even identify one a loop between helices G and H that is not possible to
detect from the hydrophobicity plot shown in Fig. 2C.
The two loops that are not identified by the reporter fusions are located between
the E-F and P-Q helices. They are both oriented towards the periplasm in the 3D
-6-
structure, but the reporter fusions made immediately downstream of the E and P helices
are cytoplasmic. In contrast to all the other Nin-Cout transmembrane helices in YadQ, the
E and P helices are thus unable to promote translocation of the reporters to the periplasm.
Interestingly, the E helix (RWWRVLPVKFFGGLGTLGG) has a rather low average
hydrophobicity, and the short P helix (APLTGIILVLEMT) is not even discernible on the
hydrophobicity plot, Fig. 2C, suggesting the possibility that the E-F and P-Q segments
insert into the membrane not as individual helices but as pre-formed “helical hairpins”
(Engelman and Steitz 1981). The E-F and P-Q hairpins are highlighted in Fig. 2A: they
are both located on the perimeter of the YadQ monomer, and it does not seem
unreasonable to assume that they can fold into pre-formed pockets in the structure only
after most or all of the other helices have assembled. This would be analogous to the
suggested late insertion of the so-called pore helices in certain potassium channels
(Umigai et al. 2003), except that the E-F and P-Q hairpins are lipid-exposed to a much
greater degree than the potassium channel pore helices. Since the P-Q hairpin is at the
monomer-monomer interface, dimer formation would only be possible after the proper
assembly of this hairpin in the monomer.
Finally, we note that when TMHMM is constrained with a more realistic set of
reporter fusions – one that it could conceivably have been constructed without
knowledge of the 3D structure – only 4 of the 7 helical hairpins are correctly predicted,
Fig. 2B (bottom). Although the hydrophobicity plot (Fig. 2C) might have led one to
suspect additional transmembrane helices in the region corresponding to helices E-G and
perhaps P-Q, it would have been very difficult to obtain strong evidence for their
existence using only reporter fusions. Possibly, complementing techniques such as Cyslabelling (van Geest and Lolkema 2000) might have helped, but it is nevertheless clear
that it is going to be very difficult to avoid artifactual results in cases where a
transmembrane helix can only insert across the membrane as part of a helical hairpin.
-7-
Given that YadQ appears to be a rather extreme case compared to most other known
membrane protein structures, this may not be too much of a problem, however.
-8-
Materials and methods
DNA techniques. The cloning of the E. coli yadQ into phoA and gfp fusion vectors was
done as described (Rapp et al. 2004; Daley et al. 2005). Primers were obtained from
Cybergene (Stockholm) or MWG (Ebersberg, FRG). All constructs were checked by
plasmid sequencing (BM labbet, Furulund, Sweden).
Experimental determination of C-terminal location. Constructs encoding PhoA
fusions were transformed into E. coli CC118 (Lee and Manoil 1994) and those encoding
GFP fusions were transformed into E. coli BL21(DE3)pLysS. The PhoA and GFP assays
were performed as described (Rapp et al. 2004; Daley et al. 2005). The assays were
repeated 3 times for each construct and the results were averaged.
Acknowledgements
The work was supported by grants from the Swedish Research Council, the Marianne
and Marcus Wallenberg Foundation, the Swedish Foundation for Strategic Research, and
the Swedish Cancer Foundation to GvH. We thank Karin Melén for help with the
TMHMM predictions.
-9-
References
Boyd, D., Traxler, B., and Beckwith, J. 1993. Analysis of the topology of a membrane
protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol 175:
553-556.
Chen, C.P., Kernytsky, A., and Rost, B. 2002. Transmembrane helix predictions
revisited. Protein Sci 11: 2774-2791.
Claros, M.G., and von Heijne, G. 1994. TopPred II: An improved software for membrane
protein structure prediction. CABIOS 10: 685-686.
Daley, D.O., Rapp, M., Granseth, E., Melén, K., Drew, D., and von Heijne, G. 2005.
Global topology analysis of the Escherichia coli inner membrane proteome. Science 308:
1321-1323.
Drew, D., Sjöstrand, D., Nilsson, J., Urbig, T., Chin, C.N., de Gier, J.W., and von Heijne,
G. 2002. Rapid topology mapping of Escherichia coli inner-membrane proteins by
prediction and PhoA/GFP fusion analysis. Proc Natl Acad Sci USA 99: 2690-2695.
Dutzler, R., Campbell, E.B., Cadene, M., Chait, B.T., and MacKinnon, R. 2002. X-ray
structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion
selectivity. Nature 415: 287-294.
Engelman, D.M., and Steitz, T.A. 1981. The spontaneous insertion of proteins into and
across membranes: The helical hairpin hypothesis. Cell 23: 411-422.
Engelman, D.M., Steitz, T.A., and Goldman, A. 1986. Identifying nonpolar transbilayer
helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem
15: 321-353.
Feilmeier, B.J., Iseminger, G., Schroeder, D., Webber, H., and Phillips, G.J. 2000. Green
fluorescent protein functions as a reporter for protein localization in Escherichia coli. J
Bacteriol 182: 4068-4076.
Kim, H., Melén, K., and von Heijne, G. 2003. Topology models for 37 Saccharomyces
cerevisiae membrane proteins based on C-terminal reporter fusions and prediction. J Biol
Chem 278: 10208-10213.
Lee, E., and Manoil, C. 1994. Mutations eliminating the protein export function of a
membrane-spanning sequence. J Biol Chem 269: 28822-28828.
- 10 -
Manoil, C. 1991. Analysis of membrane protein topology using alkaline phosphatase and
ß-galactosidase gene fusions. Methods in Cell Biol. 34: 61-75.
Manoil, C., and Beckwith, J. 1986. A genetic approach to analyzing membrane protein
topology. Science 233: 1403-1408.
Melén, K., Krogh, A., and von Heijne, G. 2003. Reliability measures for membrane
protein topology prediction algorithms. J Mol Biol 327: 735-744.
Rapp, M., Drew, D.E., Daley, D.O., Nilsson, J., Carvalho, T., Melén, K., de Gier, J.W.,
and von Heijne, G. 2004. Experimentally based topology models for E. coli inner
membrane proteins. Prot Sci 13: 937-945.
Umigai, N., Sato, Y., Mizutani, A., Utsumi, T., Sakaguchi, M., and Uozumi, N. 2003.
Topogenesis of two transmembrane type K+ channels, Kir 2.1 and KcsA. J Biol Chem
278: 40373-40384.
van Geest, M., and Lolkema, J.S. 2000. Membrane topology and insertion of membrane
proteins: Search for topogenic signals. Microbiol. Mol. Biol. Rev. 64: 13-33.
von Heijne, G. 1992. Membrane protein structure prediction - Hydrophobicity analysis
and the positive-inside rule. J Mol Biol 225: 487-494.
- 11 -
Table 1
Reporter fusion sites and activities. PhoA and GFP activities (mean of 3 experiments)
were normalized to the most active reporter fusion. Reporter fusions were classified as
- (little or no activity), + (medium activity), and ++ (high activity).
Fusion site
<PhoA>
<GFP>
PhoA class
GFP class
73
1.00
0.01
++
-
107
0.00
0.78
-
++
122
0.00
1.00
-
++
146
0.01
0.44
-
++
170
0.00
0.21
-
++
192
0.50
0.01
++
-
213
0.00
0.07
-
+
250
0.15
0.04
+
-
288
0.00
0.00
-
-
308
0.36
0.01
++
-
318
0.45
0.01
++
-
328
0.60
0.00
++
-
357
0.01
0.17
-
+
384
0.20
0.03
++
-
403
0.00
0.15
-
+
420
0.00
0.05
-
+
442
0.00
0.15
-
+
458
0.00
0.22
-
++
473
0.00
0.22
-
++
- 12 -
Figure legends
Figure 1
The ClC chloride channel protein YadQ. (A) Amino acid sequence. The 14
transmembrane helices are underlined. Boxed residues show the position of the last
residue in the YadQ part of each fusion. (B) Topology derived from the 3D structure.
Transmembrane helices are indicated by upper case letters, other helices by lower case
letters. Qualitative reporter fusion activities are shown as (-, +, ++; upper sign = PhoA,
lower sign = GFP). (C) 3D structure of the YadQ homodimer (PDB-code 1KPK). The
two subunits are shaded in gray and white. The periplasmic side is up.
Figure 2
Topology mapping results. (A) Reporter fusion activities mapped onto the 3D structure
of the YadQ homodimer (left subunit). White CPK residues indicate fusion sites with
high PhoA and low GFP activity; black CPK residues indicate sites with low PhoA and
high GFP activity. Arrows point to the reporter fusion sites in the E/F and P/Q loops. The
E-F and P-Q helical hairpins are shown as cartoons (right subunit). (B) TMHMM
predictions. The unconstrained TMHMM prediction is shown on top, the fully
constrained prediction in the middle, and the model obtained when using the fusions
indicated by arrows in panel C as constraints is shown on the bottom. The
transmembrane helices identified in the 3D structure are indicated by thin black lines. (C)
Hydrophobicity plot produced by TopPred (von Heijne 1992; Claros and von Heijne
1994) using the GES hydrophobicity scale (Engelman et al. 1986) and default
parameters. The helices identified in the 3D structure are indicated (transmembrane
helices in upper case). The reporter fusion sites are indicated by white (high PhoA, low
GFP activity) or black (low PhoA, high GFP activity) circles. Arrows point to reporter
- 13 -
fusion sites that might have been used had the 3D structure not been known (see text).
The dashed lines indicate the default cutoff hydrophobicity used by TopPred for
predicting “putative” and “certain” transmembrane helices.
- 14 -
E
F
H
I
K
N
O
Q
Figure 1A
ALLAASI R APLTGIILVLEMTDNW Q LILPMIITGLGATLLAQFTGG K PLWSAILARTLAKQE A EQLARSKAASASEN T
P
F N LIPIATAGN F SMGMLVFIFVARVITTLLCFSSGAPGGI F APMLALGTVLGTAFGMVAVELFPQWH L EAGTFAIAGMG
M
LIDVGKLSDAPL N TLWLWLILGIIFGIFGPIFNKWVLGMQDLLHRVHGGN I TKWVLMGGAIGGLCGLLGF V APATSGGG
J
GRMVLDIFRLK G DEARHTLLATGAAAGLAAAFN A PLAGILFIIEEMRPQFRWTL I SIKAVFIGVIMSTIMWRIFNHEVA
G
TVAFLCSAVLAMFGWFLVRKWAPEAGG S GIPEIEGALEDQRP V RWWRVLPVKFFGGLGTLGGGMVL G REGPTVQIGGNI
C
MKTDTPSLETPQAARLRRRQLIRQLLERDKTPLAILFMAAVVGTLVGLAAVAFDKGVAWLQNQRMGALVHTA D NWPLLL
B
a
B
++
- d
++
Figure 1B
E
++
C
++
F
++
G
++
-
+
I
J
cytoplasm
H
+
-
periplasm
-
K
M
+
++
++
- l N
++
-
+
O P
+
++
+
r
Q
Figure 1C
Figure 2A
P-Q
E-F
Figure 2B
Figure 2C
a
B
C
d
E
F
G H
I
J
K
l
M
N
O P Q
r