Download Dual-topology membrane proteins Escherichia coli Susanna Seppälä

Dual-topology membrane proteins
in Escherichia coli
Susanna Seppälä
©Susanna Seppälä, Stockholm 2011
ISBN 978-91-7447-351-3, pp. 1-66
Printed in Sweden by US-AB, Stockholm 2011
Distributor: Department of Biochemistry and Biophysics, Stockholm University
ii
Vanhemmilleni
iii
List of publications
Primary publications
I
Rapp M*, Granseth E*, Seppälä S, von Heijne G (2006):
Identification and evolution of dual-topology membrane proteins.
Nature Structural and Molecular Biology 13, 112-116
II
Rapp M*, Seppälä S*, Granseth E, von Heijne G (2007): Emulating
membrane evolution by rational design. Science 315, 1282-1284
III
Seppälä S, Slusky JS, Lloris-Garcerá P, Rapp M, von Heijne G
(2010): Control of membrane topology by a single C-terminal residue.
Science 328, 1698-1700
IV
Lloris-Garcerá P, Bianchi F, Slusky JSG, Seppälä S, Daley DO, von
Heijne G (201x): Antiparallel dimers of the small multidrug-resistance
protein EmrE are more stable than parallel dimers. Manuscript in
preparation
(* these authors contributed equally)
Additional publications
Granseth E, Seppälä S, Rapp M, Daley DO, von Heijne G (2007):
Membrane protein structural biology – how far can the bugs take us?
(Review) Molecular Membrane Biology 24, 329-332
Xie K, Hessa T, Seppälä S, Rapp M, von Heijne G, Dalbey R (2007):
Features of transmembrane segments that promote the lateral release from
the translocase into the lipid phase. Biochemistry 46, 15153-15161
Cassel M, Seppälä S, von Heijne G (2008): Confronting fusion-protein
based membrane protein topology mapping with reality: the Escherichia coli
ClcA H+/Cl- exchange transporter. Journal of Molecular Biology 381, 860866
iv
Abstract
Cellular life, as we know it, is absolutely dependent on biological
membranes; remarkable superstructures made of lipids and proteins. For
example, all living cells are surrounded by at least one membrane that
protects the cell and holds it together. The proteins that are embedded in the
membranes carry out a wide variety of key functions, from nutrient uptake
and waste disposal to cellular respiration and communication. In order to
function accurately, any integral membrane protein needs to be inserted into
the cellular membrane where it belongs, and in that particular membrane it
has to attain its proper structure and find partners that might be required for
proper function. All membrane proteins have evolved to be inserted in a
specific overall orientation, so that e.g. substrate-binding parts are exhibited
on the ‘right side’ of the membrane. So, what determines in which way a
membrane protein is inserted? Are all membrane proteins inserted just so?
The focus of this thesis is on these fundamental questions: how, and when, is
the overall orientation of a membrane protein established? A closer look at
the inner membrane proteome of the familiar gram-negative bacterium
Escherichia coli revealed a small group of proteins that, oddly enough,
seemed to be able to insert into the membrane in two opposite orientations.
We could show that these dual-topology membrane proteins are delicately
balanced, and that even the slightest manipulations make them adopt a fixed
orientation in the membrane. Further, we show that these proteins are
topologically malleable until the very last residue has been synthesized,
implying interesting questions about the topogenesis of membrane proteins
in general. In addition, by looking at the distribution of homologous proteins
in other organisms, we got some ideas about how membrane proteins might
evolve in size and complexity. Structural data has revealed that many
membrane bound transporters have internal, inverted symmetries, and we
propose that perhaps some of these proteins derive from dual-topology
ancestors.
v
Table of Contents
List of publications ...................................................................................... iv
Abstract ......................................................................................................... v
Abbreviations ............................................................................................. viii
Introduction................................................................................................... 9
The model organism ........................................................................... 10
Biological membranes ................................................................................ 12
Membrane lipids................................................................................. 13
The lipid bilayers of E. coli ................................................................ 14
The outer lipid bilayer........................................................... 14
The inner lipid bilayer........................................................... 15
Membrane proteins............................................................................. 15
Membrane protein topology.................................................. 16
β-barrel membrane proteins ................................................. 17
α-helical membrane proteins ................................................ 18
E. coli membrane proteins.................................................................. 18
Outer membrane proteins ..................................................... 18
Inner membrane proteins ...................................................... 19
α-helical membrane proteins: topology, structure and evolution.......... 20
Topology and structure of α-helical bundles ..................................... 20
Structural repeats and evolution........................................... 21
Unusual topologies ............................................................................. 23
Bona fide dual-topology membrane proteins........................ 23
Other modes of dual topology............................................... 27
Biogenesis of α-helical membrane proteins.............................................. 28
Targeting and integration of membrane proteins in E. coli................ 29
vi
Targeting of exported proteins.............................................. 29
Targeting of cytoplasmic membrane proteins....................... 29
The Sec translocon ................................................................ 30
YidC....................................................................................... 31
Topogenesis........................................................................................ 32
The nature of the polypeptide chain ................................................... 32
Hydrophobicity and aromatic amino acid residues.............. 32
Charged amino acid residues ............................................... 33
The importance of context: neighbouring helices................. 35
Role of the translocon/insertase.......................................................... 36
The size of the protein-conducting channel .......................... 36
The surrounding membrane................................................................ 37
The effect of lipids ................................................................. 37
Protein content of the membrane .......................................... 37
Methods and publications .......................................................................... 39
The model protein............................................................................... 39
Major experimental methods.............................................................. 40
Topology mapping using reporter proteins .......................... 40
Protein expression and selective radiolabelling................... 41
In vivo ethidium toxicity assays ............................................ 42
Blue-Native PAGE ................................................................ 43
Cysteine labelling and crosslinking ...................................... 43
Summary of papers............................................................................. 44
Paper I................................................................................... 44
Paper II ................................................................................. 44
Paper III................................................................................ 45
Paper IV ................................................................................ 45
Conclusions and perspectives .................................................................... 47
Populärvetenskaplig sammanfattning på svenska................................... 49
Acknowledgements ..................................................................................... 50
References.................................................................................................... 52
vii
Abbreviations
CL
cryo-EM
C-terminus
GFP
IPTG
N-terminus
SMR
PCC
PE
PG
PhoA
RNC
SRP
cardiolipin
cryo-electron microscopy
carboxy-terminus
green fluorescent protein
isopropyl β-D-thiogalactopyranoside
amino-terminus
small multidrug resistance
protein-conducting channel
phosphatidylethanolamine
phosphatidylglycerol
alkaline phosphatase
ribosome:nascent chain complex
signal recognition particle
Amino acids
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
viii
Ala
Cys
Asp
Glu
Phe
Gly
His
Ile
Lys
Leu
Met
Asn
Pro
Gln
Arg
Ser
Thr
Val
Trp
Tyr
Alanine
Cysteine
Aspartate
Glutamate
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
Leucine
Methionine
Asparagine
Proline
Glutamine
Arginine
Serine
Threonine
Valine
Tryptophan
Tyrosine
Introduction
Cellular identity relies on the existence of the cellular membrane, a
semipermeable barrier that encloses any cell and defines its boundary. In
many cells, the interior is further divided into membrane enclosed
compartments with specialized functions (organelles), and multicellular
organisms are, simply put, large conglomerates of specialized, yet discrete,
cells. The generation and maintenance of intracellular and organellar
disparity is largely managed by membrane proteins that permit a controlled,
continuous, transmembrane flow of material and information. Figuratively
speaking, membrane proteins are the windows and doors of the cell: acting
as receptors, connectors, channels and pumps, they are involved in
innumerable translocations and signalling pathways; they are crucial to cell
division and communication, as well as for energy harvesting processes such
as photosynthesis and cellular respiration. Their importance is reflected by
the fact that about a quarter of the genes in a typical organism encode
integral membrane proteins (1-3); and that the majority of marketed drugs
are, in one way or another, targeted towards membrane bound transporters,
receptors and enzymes (4, 5).
In any living cell, at any given time, a wide variety of proteins are targeted
to, and integrated into the membranes where they belong, and in those
membranes the proteins assemble into functional units. Importantly,
membrane proteins are believed to contain inherent information that is
decoded by the membrane integration machineries, and that ensures that the
protein is inserted in a correct overall orientation relative to the membrane.
This thesis examines how, and when, the correct overall orientation of
membrane proteins are established.
Most of the research for this thesis has been done using Escherichia coli
EmrE as a model protein. Therefore, much of what is said below is focused
on processes that have been observed in that particular bacterium. Naturally,
examples from, and comparisons to, other organisms are drawn and made
when deemed necessary. Here follows a short presentation of E. coli, and an
introduction to biological membranes and membrane proteins. Then follows
9
more detailed discussions on the topology and biogenesis of α-helical
membrane proteins. In an attempt to put this thesis work in a context, major
results are presented and discussed throughout. Specific comments on
methods, and a summary of the papers, are given in a separate chapter,
followed by some conclusions and perspectives.
The model organism
E. coli is a rod-shaped, ~0.5 µm wide and ~2 µm long, gram-negative
bacterium. It is part of the normal gut flora in humans and other animals,
although there are pathogenic variants that may cause disease. Since its
discovery by Theodor Escherich in 1885, E. coli has become a popular
workhorse in laboratories worldwide, the main reasons being that the cells
are easy to cultivate, and that their genetic content is easily manipulated.
Many fundamental cellular processes and pathways were first illuminated
with the aid of this bacterium, and it has proven central to our current
understanding of basic biochemistry, molecular genetics and structural
biology. It is exceptionally useful as an expression host for heterologous
proteins, not the least evidenced by the successful expression of membrane
proteins for structural studies (6, 7).
With this in mind, it is important to realize that as a taxon, E. coli comprises
strains that may differ in as much as 80% of their genes (8, 9). Typically,
E. coli cells have one circular chromosome of ~4.5 Mbp, encoding ~4500
proteins. Some strains, such as the enterotoxigenic variants, carry extrachromosomal genes on plasmids (10). Strikingly, the pan genome of
61 sequenced E. coli strains was shown to encompass ∼15 000 gene families,
and only about 1000 of these were found in every genome (9). This diversity
may be explained by frequent horizontal gene transfer events, accompanied
by a reasonably conserved chromosome size, meaning that apart from some
core genes that seem to be indispensable, the genomic material of E. coli is
highly dynamic and exchangeable (11).
The common laboratory strains derive from so called K- and B-strains that
were in use already in the early twentieth century (12-14). Most of the work
for this thesis was done using B-strain BL21(DE3) (15). The first complete
E. coli genome to be published was that of K-12 MG1655, and the recent
sequencing of the complete genome of BL21(DE3) explained interesting
differences between K- and B-strains (14, 16, 17). For example, due to
10
deletions in genes that encode flagellar components, BL21(DE3) cells do not
have flagella, and insertions in other genes cause the lack of a capsule
polysaccharide, and a truncated core oligosaccharide in the cell wall (17,
18).
Henceforth, E. coli, refers to a ‘typical’ laboratory strain, however
differences between strains are pointed out whenever necessary. As any
gram-negative bacterium, E. coli has two membranes enveloping the
cytoplasm. In between the outer and inner membranes is the periplasmic
space that contains a layer of peptidoglycan: a netlike structure made of
glycan strands that are cross-linked by peptides. In essence, the
peptidoglycan functions as an elastic net bag that protects the cells against
turgor pressure and rupture (19). The membranes and membrane constituents
are described in more detail below.
11
Biological membranes
Biological membranes have two major components: lipids and proteins. In a
seminal paper published in 1972, Singer and Nicholson conceptualized a
biological membrane as an essentially two-dimensional fluid mosaic, with
membrane proteins embedded in a lipid matrix (figure 1a) (20). Importantly,
the model recognized that proteins can move laterally in the membrane, and
associate with other membrane proteins. Indeed, it has become increasingly
clear that biological membranes are rather organized structures,
characterized by extensive interactions between a wide variety of lipids and
proteins, the latter often found in large homo- and hetero-oligomeric
complexes (figure 1b) (21).
Figure
1.
A
biological
membrane
consists
of
lipids
and
proteins.
1a)
The
classical
mosaic
model,
from
1972
(20).
1b)
A
more
recent
take
on
the
mosaic
model,
from
2005
(21).
Both
reprinted
with
permission.
Most cellular membranes contain approximately equal amounts of lipid and
protein by mass, although the ratios vary between different membranes, as
does of course the lipid and protein species. For example, the cytoplasmic
membrane of E. coli is densely packed with hundreds of different
transporters, receptors and enzymes, while specialized human myelin
sheaths are mostly lipidic and accommodates only a few different proteins
12
(22). Further, as is revealed by the variable shapes of cells and organelles,
biological membranes are highly dynamic and exceptionally flexible.
Depending on circumstances, cellular membranes can undergo considerable
qualitative changes, and they can fuse and disconnect, disassemble and
reassemble, on cue (23).
Cellular membranes function as semipermeable barriers that protect cells
and allow a controlled separation of ions, molecules and biochemical
reactions into confined spaces. The selective permeability is largely
accounted for by membrane proteins, whereas the lipids represent an
efficient barrier to most hydrophilic substances. It is however clear that the
lipids are an active part of the functional membrane; they play pivotal roles
in vesicular transport and signalling and, as is discussed below, they
influence the topology, stability and function of many membrane proteins
(23, 24). Here is a brief description of the lipid framework of a typical
biological membrane, followed by an introduction to the proteins that reside
therein.
Membrane lipids
With the exception of archaeal monolayers, a typical biological membrane is
based on two layers of lipid molecules. Common membrane lipids are
phospholipids, galactolipids, sphingolipids and sterols; their most
conspicuous feature being an extended hydrophobic part carrying a
hydrophilic headgroup (25, 26). Membrane lipids are orientated so that the
hydrophobic parts make up the bilayer core, while the polar headgroups face
the aqueous environment, forming the interfacial region. The membranes
are generally in a fluid state, but in spite of the high thermal disorder, some
structural information has been obtained, e.g. by the study of liquid
crystals of dioleoylphosphatidylcholine (27). Biological membranes are
approximately 50 Å thick, with the fatty acid core accounting for the central
∼30 Å. The polarity of the bilayer varies dramatically along the membrane
normal, from the polar interfaces to the highly hydrophobic interior, and this
creates an efficient barrier to the flow of ions and other polar molecules.
Many membrane lipids, such as the lipids in the cytoplasmic membrane of
E. coli, have a backbone of glycerol with two ester-linked acyl chains and a
phosphodiester-linked polar group. Depending on length and degree of
13
saturation, the acyl chains contribute to membrane thickness and viscosity,
and it has been shown that this trait can be conditionally altered: for
example, E. coli cells growing at room temperature have a higher degree of
unsaturated fatty acids compared to fellow cells growing at 37 °C (28). The
polar headgroups vary with respect to size and charge, and taken together,
the acyl chains and the headgroup define the overall shape of the lipid
molecule. Cylindrical lipids have similar cross-sectional areas for the
headgroups and the acyl chains, and form bilayers in aqueous solutions. In
contrast, lipids with comparatively small, or large, headgroups are conical
and prefer hexagonal phases. Both bilayer and nonbilayer prone lipids are
found in all biological membranes, and it is conceivable that just the right
mixture of lipids is necessary for maintaining membrane shape and integrity
(23, 29). In particular, the lipid composition is important for the stability and
function of many membrane proteins, for reviews see (24, 30, 31).
The lipid bilayers of E. coli
E. coli cells have two membranes: an outer membrane and an inner,
cytoplasmic, membrane. Here is a description of the lipid bilayers of E. coli,
while the proteins that are embedded in the membranes are described later.
The outer lipid bilayer
The lipid distribution in the outer membrane is asymmetric. The outer leaflet
consists of lipopolysaccharide, typically made of lipid-A molecules that are
linked, via core oligosaccharides, to highly variable O-antigen
polysaccharides (32, 33). O-antigen polysaccharides often cover the outer
surface of bacteria, however many laboratory strains lack O-antigen, and
some strains, such as BL21(DE3), have a truncated core oligosaccharide (12,
17, 18). This additional defect may explain the increased permeability of Bstrains to some antibiotics and toxins (34-36 and our unpublished
observations). The lipid composition of the inner leaflet resembles that of
the inner membrane.
14
The inner lipid bilayer
Both leaflets of the inner membrane contain phosphatidylethanolamine (PE,
~75%), phosphatidylglycerol (PG, 20-25%), and cardiolipin (CL, ~5%) (37,
38). PE is zwitterionic and nonbilayer prone due to a small headgroup size,
while the anionic PG and CL readily form bilayers. Remarkably, E. coli can
survive without PE, as is evident by the engineered AD93 strain that lacks
one of the enzymes in the PE synthesis pathway (39). The strain is not
entirely healthy, it requires divalent cations to grow in rich medium and the
cell division seems impaired – but the cells are nonetheless viable. Lipid
analyses give at hand that the membranes of AD93 are enriched in PG and
CL, and thus both the surface charge and the lateral pressure profile of the
membrane are altered. It has been proposed that the cations required for
growth make up for the lack of nonbilayer prone lipids, by altering the lipid
packing and perhaps also by neutralizing the negative charge (40).
Strikingly, monoglucosyldiacylglycerol, a nonbilayer prone lipid that is not
normally found in E. coli, can largely substitute for the lack of PE, and it has
been shown that the size of the lipid headgroup is critical for the function of
membrane proteins (29, 41). The altered lipid composition of AD93 has
major effects on the structural organisation of several membrane proteins, as
is discussed further in the chapter on α-helical membrane protein topology
below.
Membrane proteins
As mentioned above, membrane proteins intersperse all cellular membranes,
and carry out a salmagundi of key processes, from transport of nutrients and
waste products to signalling and cell division. It is well known, that proteins
often function in homo- or hetero-oligomeric complexes, and that they may
require cofactors such as metal ions or nucleotides for function. This is also
true for membrane proteins, as shown e.g. by recent analyses of membrane
protein complexes in E. coli (42, 43), for reviews see e.g. (44, 45). Here, for
simplicity, the word ‘protein’ is used to denote a ‘single polypeptide chain’,
and while allowing this, it is of course important to bear in mind that the
functional unit may comprise several polypeptide chains and other
molecules, as well as e.g. metal ions.
15
Membrane proteins are, by definition, more or less tightly associated with
membranes. Depending on the strength of the attachment, the proteins are
either peripheral or integral. Peripheral membrane proteins are engaged in
relatively shallow interactions, such as hydrogen bonding with lipid
headgroups, and they are readily extracted with carbonate (46). Many of
these interactions are transient and difficult to predict theoretically. Integral
membrane proteins, on the other hand, have hydrophobic parts that are
firmly anchored in the membrane core, and they can only be extracted by the
use of detergents or organic solvents. The hydrophobic characteristic of
these proteins make them fairly easy to find by analyzing sequence data
only, and genomic analyses suggest that about a quarter of the genes in any
organism encode integral membrane proteins (1-3).
Membrane protein topology
The topology of a membrane protein describes how the polypeptide chain
traverses the membrane and, importantly, gives the relative orientations of
the transmembrane segments and the overall orientation of the protein
relative to the membrane (47). Monotopic membrane proteins do not cross
the membrane in its entirety; bitopic membrane proteins have one
transmembrane segment; and proteins with more than one transmembrane
segment are called polytopic (47). Dual-topology membrane proteins, at the
focus of this thesis, have the unusual ability to adopt both opposite overall
orientations in the membrane.
Reflecting the organization of the lipids in the bilayer, all integral membrane
proteins are amphipathic. The hydrophobic parts anchor the proteins in the
membrane core, while hydrophilic portions interact with lipid headgroups
and polar surroundings. Any protein that resides in a lipid bilayer has to
adapt to a highly anisotropic and energetically complex environment (48,
49). Pulling hydrophobic parts out of, and pushing hydrophilic parts into, the
membrane core is energetically costly, and once properly inserted, it is thus
not likely that integral membrane proteins undergo spontaneous major
reorientations. The membrane milieu favours the formation of secondary
structure, and based on the prevailing fold, membrane proteins are either
categorized as α-helical bundles, or β-barrels (figure 2). While β-barrels
have only been found in outer membranes of gram-negative bacteria,
chloroplasts and mitochondria (50, 51), α-helical bundles constitute the
16
dominating class in virtually all other cellular membranes. Here follows a
presentation of the two protein classes.
Figure
2.
Two
classes
of
membrane
proteins.
2a)
A
β‐barrel
membrane
protein
(PDB
1BXW).
2b)
An
α‐helical
membrane
protein
(PDB
1FQY).
These,
and
following,
molecular
graphics
images
were
produced
using
the
UCSF
Chimera
package
from
the
Resource
for
Biocomputing,
Visualization,
and
Informatics
at
the
University
of
California,
San
Francisco
(supported
by
NIH
P41
RR001081),
see
http://www.cgl.ucsf.edu/chimera/.
β -barrel membrane proteins
A membrane β-barrel is made of an even number of antiparallel β-strands
that are folded into a can-like structure, so that backbone hydrogen bonds are
satisfied between the β-strands (figure 2a). Known membrane β-barrels have
between 8 and 24 strands, and typically, although not always, the primary
sequence is composed of alternating hydrophilic and hydrophobic amino
acid residues, resulting in a barrel with a hydrophilic interior and
hydrophobic exterior. Many β-barrel membrane proteins function as more or
less selective transporters, while others have been implicated in membrane
anchoring and stability (52-55). Although important for the function and
integrity of gram-negative bacteria and the organelles of endosymbiotic
origin, without further ado we will now turn our focus to α-helical
membrane proteins.
17
α -helical membrane proteins
Analyses of predicted membrane proteomes give at hand that α-helical
integral membrane proteins constitute the dominating protein class in most
cellular membranes (1, 2). α-helix formation satisfies the hydrogen bonding
potential of the polypeptide backbone, and although helices can be distorted
and interrupted, they often traverse the hydrophobic membrane core (figure
2b). α-helical membrane proteins carry out a wide range of highly
specialized functions and come in different sizes. Hypothetically, polytopic
membrane proteins can have any number of transmembrane segments. In
nature, the upper limit for an individual protein seems to be around 20
transmembrane helices, however one should not forget that functional
membrane protein complexes often contain many more helices, see e.g. (56).
Interestingly, experimental mapping of the cytoplasmic membrane
proteomes of E. coli and Saccharomyces cerevisiae, revealed that polytopic
membrane proteins often have an even number of transmembrane segments,
with both N- and C-termini in the cytoplasm (57, 58). A well-known
exception to this is the heptahelical members of the G-protein coupled
receptor superfamily. These proteins are not prevalent in either E. coli or
yeast, but are predicted to make up as much as ∼5% of the human proteome
(3, 59).
The topology, structure and biogenesis of α-helical membrane proteins are
discussed in more detail in the remaining chapters of this thesis. First,
however, is a brief look at the membrane proteomes of E. coli.
E. coli membrane proteins
Outer membrane proteins
E. coli outer membrane proteins are either lipoproteins, or β-barrels.
β-barrels make up approximately 2% of the entire E. coli proteome (60).
Most β-barrel membrane proteins form porins, passive transporters that
typically permit transmembrane diffusion of molecules up to 600 Da, while
others are important for membrane stability and adhesion (52-55). Some βbarrel proteins interact with cytoplasmic membrane proteins and play
important roles in the active extrusion of toxins, as exemplified by the
AcrAB-TolC system (61, 62). A novel, and so far unusual, structural
18
arrangement was discovered in the Wza polysaccharide transporter that
forms an α-helical barrel in the outer membrane (63). Notably, all outer
membrane proteins are synthesized in the cytoplasm and transported through
the inner membrane, further through the peptidoglycan layer, and into the
outer membrane. Several components of the complex translocation/insertion
systems have been identified, although many details remain to be described
(64).
Inner membrane proteins
About a quarter of the genes in a typical E. coli cell are predicted to encode
inner membrane proteins, and they are all of the α-helical bundle type.
About 40% of the polytopic inner membrane proteins have been predicted to
function as transporters and channels, and roughly 5% are involved in
metabolic processes, signalling, and biogenesis of the cell envelope,
respectively (57, 65). The remaining ∼35% have no annotated function.
Especially smaller membrane proteins have eluded functional
characterization, although some have been shown to be involved in
signalling and stabilization of membrane protein complexes (66, 67). A
global topology analysis of the inner membrane proteome revealed that the
majority of the polytopic proteins have an even number of transmembrane
helices and both N- and C-termini in the cytoplasm (57). As is discussed
further below, a small subset of proteins in the inner membrane of E. coli are
able to insert into the membrane in two opposite orientations (Paper I, 57,
68). As described in the chapter on membrane protein biogenesis, most
proteins in the inner membrane of E. coli are inserted via the Sec pathway,
and some of them require the insertase/chaperone YidC.
19
α-helical membrane proteins: topology, structure
and evolution
Topology and structure of α-helical bundles
α-helical, integral membrane proteins have one or more helical
transmembrane segment(s), and while the architecture of an individual
membrane protein can be quite complex, some topological features are
generally applicable. As is described in the next chapter of this thesis, all
integral α-helical membrane proteins have hydrophobic stretches, and
especially the distribution of positively charged amino acid residues has
been shown to be an important topology-determining factor. In accordance
with the positive-inside rule, cytoplasmic loops of membrane proteins tend
to be enriched in lysines and arginines, and it has been shown that the
orientation of membrane proteins can be altered by manipulation of this
charge bias (69, 70). These, and other, general characteristics of membrane
proteins have been used to successfully develop topology prediction
algorithms (2, 71, 72).
In 1975, Henderson and Unwin managed to determine the three-dimensional
structure of bacteriorhodopsin by cryo-electron microscopy (cryo-EM) (73).
Ten years later, the first atomic-resolution structure of a bacterial
photosynthetic reaction centre was solved by x-ray crystallography (74). As
of 17 August 2011, 302 unique high-resolution membrane protein structures
have been solved (http://blanco.biomol.uci.edu/mpstruc). Even if one takes
into account structural variants, and low-resolution structures, merely 2% of
the ~75 000 protein structures that have been deposited in the RCSB Protein
Data Bank are of membrane proteins (75) (http://www.rcsb.org/pdb,
http://pdbtm.enzim.hu). Nevertheless, intense research is continuously
improving the methods to over-express, purify, and crystallize membrane
proteins, and the number of atomic-resolution structures is exponentially
increasing (7, 76-78).
High-resolution crystal structures have made it clear that the helices of
polytopic membrane proteins are often rather elaborately organized
20
(http://blanco.biomol.uci.edu/mpstruc), for reviews see (79, 80). Although
some proteins, such as rhodopsins, form canonical helix bundles, in many
cases the helices are distorted, not all helices span the entire membrane, and
re-entrant loops, formed by a polypeptide segment that enters and exits the
membrane on the same side, are common. Interestingly, many membrane
proteins are built of structural repeats, as described below.
Structural repeats and evolution
In light of the scarce structural data, careful sequence analyses and
hydropathy profiling have been invaluable for the classification of
membrane proteins into different families; see e.g. (81, 82). Membrane
proteins are believed to evolve through gene duplication and fusion events
(83-87). High-resolution structures have confirmed that most membrane
proteins contain structural repeats that are arranged around an approximate
symmetry axis either perpendicular to, or in the plane of, the membrane
(figure 3) (88).
Figure
3.
LacY
is
one
example
of
a
membrane
protein
with
structural
repeats.
3a)
The
three‐dimensional
structure
of
LacY,
with
the
N‐
and
C‐terminal
halves
in
different
shades
of
grey
(PDB
1PV7).
3b)
A
topology
map
of
LacY,
indicating
the
parallel
organisation
of
the
N‐
and
C‐terminal
halves
(see
text).
The
transmembrane
segments
are
represented
as
grey
and
white
sausages,
and
the
N‐
and
C‐termini
are
indicated.
Generally, protein structure is more conserved than primary sequence. Two
proteins with similar structures are not necessarily evolutionarily related, but
at the same time this implies that proteins, or protein domains, that are
related may share the same fold, yet the relationship is not necessarily
apparent at sequence level (89). This is for example the case of the E. coli
21
AmtB ammonia channel, where the relationship between the repeated
structural units is no longer visible in the primary sequence (90).
One example where the repeated elements are apparent at sequence level is
the family of the hexahelical ADP/ATP carriers. Structural data show that
these proteins are composed of three hairpin repeats that are arranged around
a pseudo-threefold axis in the membrane (91), and the tripartite architecture
was predicted by sequence analysis suggesting a triplication of the basic
hairpin element (81). Another example is the arrangement of the 12
transmembrane helices of the E. coli multidrug transporter AcrB (belonging
to the Root Nodulation and Division family). AcrB is folded in such a way
that the six N-terminal and six C-terminal helices form domains that are
related by twofold pseudo-symmetry (61). The 12 helices of E. coli Lactose
permease, LacY (Major Facilitator Transporter superfamily), are
correspondingly organized (figure 3). Again, the two halves of the protein
are folded with twofold pseudo-symmetry around the central, substratebinding cavity (92). Interestingly, each half of LacY is in turn composed of a
three-helix repeat with internal, inverted symmetry (93). Similar invertedrepeats are seen in e.g. aquaporins, the H+/Cl- exchange transporters, and the
aforementioned E. coli AmtB ammonia channel (figure 4) (90, 94, 95). The
inverted-repeats are arranged around an approximate twofold axis in the
membrane plane, and in contrast to the previous examples where the
homologous units are parallel in the membrane, the inverted domains are
oppositely orientated (96).
Figure
4.
AQP1
is
one
example
of
a
membrane
protein
with
inverted
structural
repeats.
3a)
The
three‐dimensional
structure
of
AQP1,
with
the
N‐
and
C‐
terminal
halves
in
different
shades
of
grey
(PDB
1FQY).
3b)
A
topology
map
of
AQP1,
indicating
the
antiparallel
organisation
of
the
N‐
and
C‐terminal
halves.
The
transmembrane
segments
are
represented
as
grey
and
white
sausages,
and
the
N‐
and
C‐termini
are
indicated.
22
Among the structures are many more examples of internal structural
symmetries, especially in the class of transporters, and it is believed that the
symmetrical architecture is necessary for function.
A particularly interesting structure for this thesis is that of the small
multidrug resistance (SMR) transporter EmrE from E. coli, showing an
antiparallel dimer composed of two identical, yet oppositely orientated
subunits (97). This unusual arrangement was first suggested by projection
structures determined by cryo-EM and image reconstruction of twodimensional crystals (98, 99). As is discussed below, the work presented in
this thesis further support the notion that EmrE is a genuine dual-topology
membrane protein, and as such it adopts two opposite orientations in the
membrane.
Unusual topologies
The topology of a membrane protein is sometimes dynamic and can be
conditionally altered. As previously mentioned, E. coli strain AD93 lacks the
zwitterionic membrane lipid PE, and has a negatively charged cytoplasmic
membrane owing to elevated levels of lipids PG and CL (39). The altered
lipid composition of AD93 has a fascinating effect on the topology of LacY
that exhibits a partial and reversible topological inversion (100, 101). Similar
lipid-dependent reorganisations have been shown to occur in a number of
membrane bound transporters expressed in AD93, and it seems clear that
these rearrangements are the result of a complex interplay between charges
in the lipids and in the proteins (102-104). Further, there are proteins that
exhibit somewhat unusual topological arrangements in their native
membrane, and some of them are described here.
Bona fide dual-topology membrane proteins
A dual-topology membrane protein adopts two opposite orientations in its
native membrane. A global topology analysis of the inner membrane
proteome of E. coli disclosed a group of five proteins that seemed to have
dual topology (57). A closer look at these proteins revealed that they are
small, consisting of around 110 amino acid residues folded into four
transmembrane segments, and, importantly, they have very few positively
charged amino acid residues that are evenly distributed between cytoplasmic
23
and periplasmic loops (figure 5). In other words, these proteins have a very
weak charge bias, and we could show that in compliance with the positiveinside rule, they are very sensitive to the addition or removal of positively
charged residues (Paper I). One of these proteins was the aforementioned
SMR transporter EmrE, the others were the SMR transporter SugE,
camphor-resistance protein CrcB, and two were proteins of unknown
function. Subsequent topology analyses have confirmed the dual topology of
several other SMRs (105).
Figure
5.
The
dual
topology
of
E.
coli
EmrE.
5a)
The
structure
of
the
antiparallel
homodimer,
with
the
identical
subunits
in
different
shades
of
grey
(PDB
3B5D).
5b)
The
topology
of
EmrE,
indicating
the
dual
topology
and
the
weak
charge
bias.
The
transmembrane
helices
are
represented
as
grey
sausages,
and
the
N‐
and
C‐termini
are
indicated.
The
filled
black
circles
are
positively
charged
amino
acid
residues
(K22,
R29,
R82,
R106).
As mentioned above, the dual topology of EmrE is supported by structural
data (97-99). The high-resolution crystal structure is readily superimposed
onto the cryo-EM density map (97). Importantly, it has been shown that the
two-dimensional crystals bind substrate in a site between two monomers,
with affinities that are indicative of a functional protein (99, 106-108).
Cysteine labelling has further validated the dual orientation of EmrE
monomers (109, 110). Taking into account evolutionary constraints and the
position of essential residues (111-113), a model was based on the cryo-EM
structure that supports antiparallel organisation of the subunits in the dimer
(114). However while it seems clear that monomeric EmrE is a genuine
dual-topology membrane protein, the relative orientation of the subunits in
the functional dimer is under debate (Paper IV, 109, 115-118).
Other SMRs have been shown to consist of obligate heterodimers, such as
E. coli YdgEF (MdtJI), and YkkCD and EbrAB from Bacillus subtilis (11924
121). The subunits of the dimeric EbrAB have opposite orientations in the
membrane, and normally both are required for function (122). By
exchanging and shortening the loops of the proteins, Kikukawa and coworkers managed to generate solely functional EbrA and EbrB (123, 124).
The changes made, were in fact a manipulation of the distribution of
positively charged amino acid residues, and seeing that the wild type protein
forms an antiparallel dimer it is feasible that the solely functional proteins
have dual topology, so that the antiparallel organisation is maintained.
A case of putative dual topology was found by sequence analyses of
DUF606, a bacterial membrane protein family of unknown function. This
family comprises i) genes that are predicted to encode dual-topology
membrane proteins, ii) paired genes that encode homologous proteins that
have a fixed orientation in the membrane, and iii) genes that encode large
membrane proteins with homologous, oppositely orientated halves (Paper I,
85). The presence of these three topological variants in the same family
suggests underlying evolutionary pathways, where a gene encoding a dualtopology protein can undergo duplication and/or fusion to generate
homologous, oppositely orientated proteins, and proteins with structural,
inverted, repeats (figure 6) (85).
The E. coli glutamate transporter GltS is a ten-transmembrane helix protein
that is predicted to have an inverted structural repeat (125). In a study of the
evolution of antiparallel two-domain membrane proteins, GltS was split,
whereafter the two halves of the protein were fused in the reverse order,
without loss of function in any case (126). On the same note, we duplicated
the gene encoding EmrE, and made changes in the genes so that they
encoded proteins with fixed, opposite, orientations in the membrane (Paper
II). Importantly, the two oppositely orientated EmrE variants had to be coexpressed in order to get a functional transporter.
25
Figure
6.
Proposed
evolutionary
relationships
between
dual‐topology
and
inverse‐topology
membrane
proteins.
Gene
X
encodes
a
dual‐topology
membrane
protein,
and
undergoes
duplication/fusion
events.
Genes
Xa
and
Xb
encode
homologous,
oppositely
orientated
membrane
proteins,
while
gene
Xa‐
Xb
encodes
a
twice
as
large
membrane
protein
with
an
inverted
structural
repeat.
Reprinted
with
permission
from
(127).
Recently, experimental topology mapping of a number of small proteins in
the cytoplasmic membrane of E. coli suggested that some of these onetransmembrane segment proteins exhibit dual topology (68). The function of
these proteins is not known. Another one-transmembrane segment protein is
MRAP, the Melanocortin 2 receptor accessory protein that is involved in the
trafficking of the G-protein coupled receptor MC2 to the plasma membrane
in adrenal glands. The protein functions as a homodimer and the two
subunits have opposite orientations in the membrane (128). As such, it is the
first eukaryotic antiparallel homodimer that has been reported to date.
Further analyses indicated that antiparallel dimerisation is a prerequisite for
the function of MRAP, and that the proteins are synthesized in two opposite
orientations, i.e. the final antiparallel topology is acquired in the
endoplasmic reticulum (129).
26
Other modes of dual topology
Ductin is a four-transmembrane helix protein that does not only exhibit dual
topology, but also dual function (130). However it is worth to notice, that in
this case, the two opposite topologies - and functions - exist in different
cellular membranes. A hexamer of ductins make up the vacuolar Vo, which
is a part of the vacuolar V-ATPase. In this scenario, ductin adopts a topology
where both its N- and C-termini are in the extracytoplasmic lumen.
However, ductin is also a part of a connexon channel in gap junctions, and
there it has the opposite topology, i.e. both N- and C-termini are in the
cytoplasm. How this dual topology has evolved is not clear.
Yet another example of dual topology is seen in the Hepatitis B virus large
envelope protein (131). At the endoplasmic reticulum, the protein is inserted
with a three-transmembrane helix topology. During maturation, in
approximately 50% of the molecules, an N-terminal segment is inserted to
generate a four-transmembrane segment topology. This mixed arrangement
is preserved in the viral envelope, and presumably the two topologies
represent two different functions.
The structures and topologies that are described here are just a few examples
of what α-helical membrane proteins look like. Further experimentation and
the increasing number of high-resolution crystal structures are likely to
reveal additional cases of ‘odd’ structural arrangements that may be difficult
to predict from sequence. These, and the examples mentioned previously,
show that while topology can be complicated and dynamic, there are a few
basic features that seem all-important for topogenesis, such as
hydrophobicity and charge distribution. So, how are membrane proteins
inserted into the membranes? What are the topology determinants? These
questions are addressed in the next chapter of this thesis.
27
Biogenesis of α-helical membrane proteins
Most membrane proteins are integrated in a co-translational manner, and
cellular insertion machineries have evolved to handle a number of
challenging tasks: how are proteins targeted to the membrane? How are
transmembrane segments recognized and inserted? How do transmembrane
segments interact with each other, and how is the correct composition and
stoichiometry of protein complexes controlled? Central to this thesis is the
fundamental question of orientation: how, and when, is the correct topology
of a membrane protein established?
With the exception of a few proteins encoded by mitochondrial and
chloroplast genomes, cellular proteins are synthesized by cytoplasmic
ribosomes. Proteins that exhibit their function in specific compartments, or
outside the cell, are targeted to their respective locations either co- or posttranslationally, as suggested by Blobel and coworkers some 40 years ago
(132, 133). Bacterial and archaeal cells have machineries that allow targeting
of proteins to, into, and through the cellular envelope; and eukaryotic cells
have additional elaborate systems that ensure that nuclear, mitochondrial,
chloroplast, and other organellar proteins end up in their respective
compartment, see e.g. (134-138).
The cytoplasmic membrane integration process in E. coli is similar to the
processes taking place at the cytoplasmic membrane of archaea, and at the
endoplasmic reticulum of eukaryotes, and many of the participating
molecules are homologous and universally conserved (139). Here is a
presentation of the cytoplasmic membrane protein insertion systems in
E. coli, followed by more general discussions on membrane protein
topogenesis.
28
Targeting and integration of membrane proteins in E. coli
Targeting of exported proteins
In E. coli, proteins are targeted to the cytoplasmic membrane either for
insertion or for translocation, for recent reviews see (135, 139, 140). Most
exported proteins contain a well-characterized N-terminal cleavable signal
sequence (141), and are targeted to the membrane via the post-translational
SecB-pathway (142). SecB is a cytoplasmic chaperone that interacts with the
exported protein, keeping it in a translocation compatible, unfolded, state.
The substrate is delivered to the membrane, and facilitated by the SecA
ATPase, the protein is fed through the membrane-embedded and universally
conserved Sec translocon (143). Proteins that are translocated in a folded
form, perhaps because cofactors have to be inserted previous to export,
generally use the Twin-Arginine Translocation (TAT) system that
exclusively handles folded proteins (144, 145).
Targeting of cytoplasmic membrane proteins
Most cytoplasmic membrane proteins in E. coli are targeted to the membrane
by the universal signal recognition particle (SRP) pathway and inserted by
the Sec translocon; in addition some proteins require the insertase/chaperone
YidC for proper integration and folding, see e.g. (45). Compared to its
substantially larger mammalian homolog, the bacterial SRP is minimalistic,
comprising only a protein called Ffh and a 4.5S RNA (146, 147). When the
ribosome starts translating an mRNA that encodes a membrane protein,
perhaps already when the nascent chain is in the exit tunnel of the ribosome,
SRP binds to the ribosome:nascent chain complex (RNC) (148, 149). In
eukaryotes, it is believed that SRP binding stalls translation, but whether this
is the case in E. coli is not entirely clear (150, 151). In any case, the RNC is
guided to the membrane by SRP and its receptor, E. coli FtsY (152, 153).
The GTPase activities of SRP and FtsY now leads to the disassembly of
RNC:SRP, and subsequent docking of RNC onto the membrane embedded
protein-conducting channel. Powered by the ribosome, the nascent chain is
fed through - or into - the membrane, while it is being made.
Whether, and how, a transmembrane segment is recognized and inserted into
the membrane is the result of the interplay between several factors that will
be described below. First, however, follows a description of the major
29
known membrane protein insertion machineries in E. coli, the Sec translocon
and the YidC insertase/chaperone.
The Sec translocon
One of the major protein translocation systems, not only in E. coli but in any
cell, is the Sec machinery. At the core of the Sec machinery is the membrane
embedded Sec translocon that allows both export of proteins through the
membrane, and integration of proteins into the membrane. The central
protein-conducting/inserting channel (PCC) is found in the cytoplasmic
membranes of bacteria (SecYEG) and archaea (SecYEβ), and in the
endoplasmic reticulum of eukaryotic cells (Sec61αβγ) (139). SecYE/αγ are
homologous and form the core of the channel, while the G/β subunits are
neither homologous nor essential. Parts of the PCC are also found in
thylakoid membranes of chloroplasts, and while most mitochondria seem to
have lost the genes, the mitochondrial genome of the unicellular fungus
Reclinomonas americana encodes a SecY homolog, corroborating that the
Sec machinery is truly universal (154, 155).
The high-resolution crystal structure of SecYEβ from Methanocaldococcus
jannaschii provided clues as to how the PCC handles the dual task of
translocation/insertion (figure 7) (156). SecY forms the actual channel, SecE
seems to form a stabilizing clamp that holds the channel together, and the
nonessential Secβ is peripherally located. SecY has 10 transmembrane
helices and viewed from the plane of the membrane, it has an hourglass
shape (figure 7a). At the most constricted point, the channel is lined by a
ring of hydrophobic amino acid residues, and in the non-translocating
structure, a re-entrant loop on the periplasmic side seems to act as a plug that
prevents uncontrolled leakage of ions from one side of the membrane to the
other. It has been suggested that when the Sec translocon is activated, the
plug moves out of the way so that polypeptide chains can pass (157).
Viewed along the membrane normal, SecY has an approximate clamshell
shape: the two halves of the protein, that incidentally are related by an
inverted twofold pseudo-symmetry, seem to be hinged around
transmembrane helices 5 and 6; and between transmembrane helices 7 and 8
there is an opening that has been suggested to function as a lateral gate
through which the polypeptide chain is released into the membrane bilayer
(figure 7b) (156).
30
Figure
7.
M.
jannaschii
SecYEβ
heterotrimer
(PDB
1RHZ).
7a)
View
from
the
membrane
plane.
SecY
and
SecE/β
are
in
different
shades
of
grey.
The
asterisk
indicates
the
lateral
gate.
7b)
Cytoplasmic
view.
It has been proposed that during translocation, the channel gate opens
towards the membrane lipids in such a way that a passing polypeptide chain
has access to the lipid environment (158-160). If the passing polypeptide is
hydrophobic enough, it will partition into the hydrophobic bilayer; otherwise
it will pass by and exit on the other side of the membrane.
In E. coli, SecY has ten, SecE has three, and SecG has two transmembrane
helices. The overall architecture of the core SecYE is conserved (161).
Interestingly, E. coli can be depleted of many components of the Sec
machinery and still survive (162-164). In addition to the components
mentioned above, the E. coli Sec machinery also includes the heterotrimeric
membrane bound SecDF-YajC complex (165). The exact role of these
proteins is not known, however it has been suggested that SecDF aids in
proton-motive force driven translocation, regulates the membrane cycling of
SecA, and mediates contact between SecYEG and YidC (166-168).
YidC
Some proteins in the cytoplasmic membrane of E. coli require YidC for
proper insertion and/or assembly (45, 169-172). YidC is an integral
membrane protein belonging to the YidC/Oxa1/Alb3 family that has
members in bacteria, mitochondria, chloroplasts and some archaea (173,
174). E. coli YidC has six transmembrane helices, however most family
members are lacking the N-terminal helix and the following periplasmic
31
loop (174). Apart from assisting in the assembly of some Sec-dependent
membrane proteins, YidC has been shown to function as an independent
insertase for a number of small, hydrophobic membrane proteins such as
M13 and Pf3 phage coat proteins (169, 175, 176). A recent global proteomic
study of YidC-depleted E. coli cells revealed that in particular, proteins with
comparatively small soluble domains (<100 amino acid residues) are
sensitive to YidC depletion (177).
The exact details of how YidC recognizes and integrates membrane proteins
are not clear. Cross-linking studies have shown that the conserved third
transmembrane helix in E. coli YidC contacts nascent membrane proteins
(178). A cryo-EM structure of YidC bound to a translating ribosome
suggests that YidC functions as a dimer (179), and a recent study indicated
that YidC-mediated insertion can be facilitated by additional membraneassociated proteins, such as YidD (180).
Topogenesis
The polypeptide chains of membrane proteins are believed to contain
inherent information that is decoded by the membrane integration
machinery, and that ensures correct insertion and overall orientation of the
protein in the membrane. How a membrane protein is inserted into the
membrane depends on several factors. Primarily, the nature of the polyptide
segment is important, with respect to hydrophobicity and distribution of
charged amino acid residues. Second, the actual insertase and associated
proteins may provide interaction surfaces that facilitate insertion and
topogenesis, and third, it is conceivable that the surrounding lipids and the
protein content of the membrane are likewise important for insertion,
assembly and oligomerisation.
The nature of the polypeptide chain
Hydrophobicity and aromatic amino acid residues
It has long been recognized, that a firm anchoring of proteins in biological
membranes requires hydrophobicity (181, 182). More recently, the free
energy of transfer from the polar interior of the translocon into the bilayer
32
membrane was systematically determined for each of the twenty naturally
occurring amino acids, and the experimental data was subsequently used to
develop and improve algorithms for membrane protein topology prediction
(72, 183, 184). The ‘molecular code’ for insertion into the endoplasmic
reticulum is similar regardless of the orientation of the helix (185). As
expected, it was shown that hydrophobic amino acid residues promote
membrane insertion of a model helix, while charged and other polar residues
oppose it (184). In a recent study, non-proteinogenic amino acids with
increasingly large hydrophobic side chains were incorporated into a similar
model helix, and it was shown that membrane partitioning is directly
proportional to the hydrophobic area of the amino acid side chain (186).
The distribution of amino acids in transmembrane helices is restricted by the
nature of the side chains, with respect to hydrophobicity, bulkiness, and
opportunities for intra- and interhelical interactions. Leu, Ile, Val, and Ala
are good helix-formers and often found in transmembrane segments, while
Pro is known to induce kinks (187-189). Gly, the smallest of the amino acids
with a side chain consisting of only one hydrogen atom, is considered a helix
breaker in soluble proteins but is often found in transmembrane helices
where it is involved in helix-helix interactions (190-192). For some cases the
position of the amino acids in the chain is important, as in the case of
tryptophans and tyrosines, and charged amino acid residues (see below).
Centrally placed Trp and Tyr oppose the insertion of a model helix, while
the same residues are permitted, or even enhance insertion, when moved
toward the ends of the helix (184). This is entirely in accordance with what
has been observed for natural proteins, where Trp and Tyr are often found in
the interfacial regions, an occurrence referred to as the aromatic belt. The
aromatic residues interact favourably with the polar headgroups of the lipids,
and are believed to anchor and fix tilt angles of the helices relative to the
bilayer (193-195).
Charged amino acid residues
Flanking charges have been shown to be important topology determinants;
with the most conspicuous example being the distribution of positively
charged amino acid residues Lys and Arg. Statistical sequence analyses
revealed that positively charged residues are especially prominent in the
cytoplasmic loops of membrane proteins, as stated by the positive-inside rule
(69). The distribution of positively charged amino acid residues has been
33
shown to act as a strong topological determinant, and seems generally
applicable (70, 187). The topology of membrane proteins can be inverted by
the addition of positively charged amino acid residues to extramembraneous
loops (Paper II, 70). Similarly, ‘frustrated’ membrane proteins where
marginally hydrophobic helices are forced to insert, or hydrophobic helices
are forced out of the membrane, have been generated by simple
manipulation of the positive charge bias (196, 197). We have shown that for
the dual-topology membrane protein EmrE, the effect of a single positive
charge is equally strong regardless of position: an N-terminally placed
positive charge fixes the orientation of the protein as well as a C-terminally
placed positive charge (Paper III).
Lysines and arginines have a similar and equally strong effect on the
topology of transmembrane proteins (198, 199). Histidines are partially
positively charged during cellular conditions and exhibit a similar
topological effect as Lys and Arg, providing that three or more His residues
are present (Paper III, 199). As topology determinants, negatively charged
residues are much less potent than positively charged residues, unless they
are present in large quantities (198). However there are some indications that
in some cases the negatively charged amino acids are important, and while
there is no conclusive evidence for a ‘negative-outside rule’, sometimes the
difference between positively and negatively charged amino acid residues
might be a more suitable determining factor, especially for eukaryotic
membrane proteins (104, 200).
Importantly, transmembrane helices are not all hydrophobic, but rather often
contain polar and even charged amino acid residues that act as functional
groups (201). One example is the multiple Arg in the S4 segment of the
KvAP voltage-dependent potassium channel; another is the highly conserved
Glu in the first transmembrane helix of SMR transporters (202-204).
Neighbouring residues and helices conceivably aid the insertion of positively
and negatively charged amino acid residues into the membrane. In addition,
Lys and Arg are able to exhibit ‘snorkelling’ with the side chains being so
long that the charged end groups may be in contact with the polar lipid-water
interface region, while the aliphatic parts of the residues remain in the
hydrocarbon region, maximizing energetically favourable interactions (205).
34
The importance of context: neighbouring helices
It is becoming increasingly clear that some polytopic membrane proteins are
inserted correctly only when the whole protein is present, and that the
individual transmembrane helices can not always be inserted on their own.
In some cases, the insertion efficiency of a transmembrane segment is
heavily affected by neighbouring helices and flanking residues, see e.g.
(206-209). The eukaryotic ABC-transporters P-glycoprotein and CFTR have
been shown to exhibit multiple topologies, likely caused by inefficient
insertion of marginally hydrophobic helices (206, 210, 211). Native
aquaporins have six transmembrane helices and while each subunit forms a
functional channel, they all form homo-tetramers in membranes. Despite
these structural, and functional, similarities aquaporins seem to display
different modes of membrane integration. The six helices of AQP4 are
inserted in an orderly fashion, while AQP1 is initially inserted in a fourtransmembrane helix topology (212, 213). During maturation, the third
transmembrane helix of AQP1 undergoes a 180° rotation, so that the loops
between helices 3-4 and 4-5 are pulled through the membrane, resulting in
the final six-membrane spanning topology. A similar case is found in the
archaeal glutamate transporter homolog GltPh from Pyrococcus horikoshii
(214). This protein functions as a homotrimer and each subunit has eight
transmembrane helices. Transmembrane helix 4 is marginally hydrophobic
and kinked, and is initially not inserted, but rather pulled into the membrane
at a later stage. The mechanisms for these rearrangements is not known,
however it is likely that they are coupled to, and stabilised by, assembly and
oligomerisation. Selective retention of transmembrane helices at the
translocon has also been seen for e.g. the heptahelical transmembrane
protein Opsin, where the three last transmembrane helices remain in the
vicinity of the translocon until the whole protein has been synthesized (215,
216). Also, as mentioned above, we could show that a C-terminally placed
positively charged amino acid residue had an effect on the overall topology
of the small multidrug transporter EmrE (Paper III). This is telling of a
remarkable topological malleability, however the underlying mechanisms
are not clear.
35
Role of the translocon/insertase
It is conceivable that the insertion machinery affects the way a membrane
protein is integrated into the membrane. A polypeptide chain that is fed into
the PCC is either laterally released into the lipid bilayer, or it remains in the
interior of the channel and undergoes subsequent translocation (156). At the
centre of the translocation channel of SecY/61α, there is a ring of six
hydrophobic residues believed to function as a seal against the flow of ions
during translocation. It has been shown in yeast, that if these residues are
replaced with polar or charged residues, insertion efficiency of a moderately
hydrophobic test segment was increased (217). This indicates that the
protein-conducting channel adjusts the hydrophobicity threshold for
membrane partitioning. Also other residues, e.g. in the plug-region, of the
SecY/61α translocon have been shown to affect the integration efficiency
and orientation of transmembrane segments (218).
How membrane proteins are fed into the PCC is not entirely clear.
Experimental data indicates that regardless of the final orientation, the first
transmembrane segment of a membrane protein inserts head-first into the
Sec61α translocon pore (219, 220). This implies that a segment that has its
N-terminus in the cytoplasm has to reorient, and it has been suggested that
the segment has a limited amount of time to do so (219). An alternative
mechanism for insertion of individual helices is the “hairpin model”, where
the polypeptide chain is looped into the protein-conducting channel, and
once sufficient length of downstream sequence is translated, the segment is
integrated into the membrane, see e.g. (221). It is not clear if all
transmembrane segments follow the same mode of insertion, and the
molecular details of reorientation and looping of transmembrane segments
remain to be described.
The size of the protein-conducting channel
In any case, the size of the actual PCC seems important. In the M. jannaschii
structure of SecY, which is believed to represent the closed state, the central
pore is narrow, and the ~10 Å diameter provides space for one
transmembrane helix at a time (156). Recently, SecYEG from E. coli was
challenged with sizable rigid spherical molecules fused to known Secdependent preproteins, and it was shown that the translocon could handle
molecules up to 22-24 Å (222). However there is fluorescence quenching
36
data indicating that the size of the eukaryotic Sec61 translocon pore is
substantially larger (40-60 Å) during co-translational translocation (223).
The oligomeric state of active SecY/61 complexes has been under debate,
however crosslinking- and structural data agree that while the Sec translocon
is capable of forming higher oligomers, a single copy of the heterotrimer is
able to form an active PCC (224-226). Lastly, the role of Sec-translocon
associated proteins should not be underestimated, as they may provide
interaction surfaces that enhance insertion and topogenesis. Examples of
such proteins are the aforementioned E. coli SecDF-YajC, and YidC; and
although not discussed in this thesis, eukaryotic cells have their own setup of
additional translocon-associated proteins, such as TRAM, that have been
implied in the insertion and assembly of membrane proteins, see e.g. (221)
and references therein.
The surrounding membrane
The effect of lipids
As discussed above, membrane lipids are important for proper insertion and
stability of many membrane proteins, for reviews see (24, 30, 31). The
aforementioned lipid-dependent topological inversions of transporters in
E. coli are striking examples (100-104). Also, anionic phospholipids have
been shown to be important for topogenesis of E. coli Leader peptidase, Lep,
in vitro (227). In eukaryotic cells, many membrane proteins are inserted into
the endoplasmic reticulum and subsequently targeted to e.g. the plasma
membrane by vesicular transport. The lipid composition of the different
membranes in a eukaryotic cell varies, and it is not unlikely that this affects
the topology, and function, of the membrane-embedded proteins.
Protein content of the membrane
Most biological membranes are quite densely packed with many different
kinds of proteins (22). It is perhaps difficult to test in vivo, but a recent
computational simulation suggests that the protein content of the membrane
is important for efficient insertion of especially polar amino acid residues
(228). In contrast to models assuming that the membrane consists of pure
lipid, this model incorporated protein segments into the bilayer. The result
37
was that the free energy cost of inserting a positively charged amino acid
residue into the membrane was dramatically lower, compared to the pure
lipid-models, and in fact similar to experimentally determined values (183,
184).
38
Methods and publications
Here is a presentation of the major experimental methods used throughout
this work, with comments. Then follows a summary of the papers that are
included in this thesis.
The model protein
A lot of the work for this thesis was done on E. coli EmrE, a secondary
transporter belonging to the SMR family (figure 8) (229). EmrE is expressed
in the cytoplasmic membrane of E. coli, and it uses the proton gradient to
extrude hydrophobic, cationic toxins such as ethidium, acriflavine, and
methyl viologen, see e.g. (230). EmrE is 110 amino acids long and has four
transmembrane helices. As indicated in figure 8, it has four positively
charged amino acid residues (K22, R29, R82, R106), one histidine (H110),
and three negatively charged amino acid residues (E14, E25, D84). The
mechanism of EmrE has been carefully studied. It has been shown that the
minimal functional unit is a homodimer, although the presence and
functional importance of higher oligomers in vivo cannot be excluded (106,
231, 232). EmrE binds toxins and protons in a site between two monomers,
and the binding is coordinated by a glutamate from each subunit (111-113).
The glutamate in this position (E14) is highly conserved within the SMR
family and cannot be replaced even by an aspartate in the wild-type protein,
however we have shown that an Asp is allowed in one of the subunits in the
functional dimer, although the function is slightly impaired (Paper II, III,
111, 112). While structural, biochemical and phylogenetic data support a
dual-topology for EmrE (Paper I, II, III, 97-99, 110), the organization of the
subunits in the dimer is still under debate (Paper IV, 109, 115-118).
39
Figure
8.
The
topology
and
function
of
E.
coli
EmrE.
An
antiparallel
EmrE
dimer
is
shown.
Transmembrane
segments
are
represented
as
grey
sausages,
and
the
N‐
and
C‐termini
are
indicated.
The
filled
black
circles
are
positively
charged
amino
acid
residues
(K22,
R29,
R82,
R106),
the
filled
grey
circles
are
histidines
(H110),
and
the
empty
circles
are
negatively
charged
amino
acid
residues
(E14,
E25,
D84).
The
antiporter
function
is
indicated.
Major experimental methods
Topology mapping using reporter proteins
One way to experimentally probe the topology of a membrane protein is to
use reporter proteins such as Green Fluorescent Protein (GFP) and Alkaline
Phosphatase (PhoA). These two reporters are particularly useful in E. coli,
because their activities are disparately localized. Most GFP-variants only
fold and become fluorescent in the cytoplasm of E. coli; while PhoA, owing
to the requirement of a couple of disulfide bonds, is active only when
localized to the periplasm (233, 234). Therefore, if GFP or PhoA is fused to
the membrane protein of interest, the localization of the fusion point
(cytoplasm/periplasm) is easily assayed. This way, GFP and PhoA have been
successfully used to determine the location of the C-terminus of virtually
every single polytopic protein in the inner membrane of E. coli, and GFP has
further proven useful for monitoring the expression and folding of
40
membrane proteins e.g. for structural studies (57, 235-237). Here, we have
used GFP and PhoA to monitor changes in the overall orientation of e.g.
EmrE-variants (Paper I, II). However adding a tag to a topologically
sensitive membrane protein may in itself affect the overall orientation, and
therefore tags should be used with care for topological studies (Paper II).
This is particularly true for tags that contain charged residues and/or
histidines (Paper III, 109). In some cases it can be desirable to do
complementary topology determinations e.g. by selective cysteine labelling,
see below.
Protein expression and selective radiolabelling
Throughout this work we have expressed and selectively radiolabelled EmrE
with 35S-methionine, using E. coli BL21(DE3) cells, pET-vectors, and the
rifampicin blocking technique (15, 238). E. coli (DE3)-strains have a gene
encoding T7 RNA polymerase in their chromosome (15). The T7 RNA
polymerase originates from bacteriophage T7, and recognizes a promoter
sequence that is not naturally present in the E. coli genome, but that is
present in commercially available pET-vectors. In the cells, the expression
of the T7 RNA polymerase is under control of the lacUV5 promoter, and
inducible by isopropyl β-D-thiogalactoside (IPTG). IPTG induction leads to
the production of T7 RNA polymerase, and to the subsequent expression of
any genes that are under control of a T7 promoter. Rifampicin is an
antibiotic that inhibits bacterial RNA polymerases, while the T7 RNA
polymerase is left unaffected (238). Therefore addition of rifampicin results
in the selective expression of T7 promoter-controlled genes, and this can be
used for selective radiolabelling: E. coli (DE3) cells are treated with IPTG
and rifampicin, and any gene(s) under the T7 promoter are subsequently
selectively labelled by the addition of 35S-methionine. The gene products are
easily analyzed by SDS-PAGE. This is particularly convenient if no
antibody is available against the protein of interest, as in the case of EmrE.
The pET Duet-1 vector (Novagen) used in this work has two multiple
cloning sites, each preceded by a T7 promoter/lac operator and a ribosome
binding site. This allows for co-expression of two genes, however while the
expression from the two cloning sites is strong, it is not equal (Paper IV). It
is worth to note that the DE3/T7-system described here is ‘leaky’ and that
some expression occurs even in the absence of inducer (239). This is of
41
course of special importance if the gene product is somehow toxic to the
cells, as is often the case with membrane proteins (77).
In vivo ethidium toxicity assays
As described above, E. coli EmrE renders the cells resistant to e.g. ethidium.
Throughout the work for this thesis, we have introduced many changes to
the primary sequence of EmrE, e.g regarding the distribution of positively
charged amino acid residues. Of course, when one changes the primary
sequence of a protein, it is important to test whether the protein is still
functional. For this purpose, we used an in vivo ethidium toxicity assay, with
liquid cell cultures or plates. In short, E. coli BL21(DE3) cells were
transformed with the relevant plasmids and grown in the presence of varying
concentrations ethidium bromide. To avoid toxic EmrE levels due to overexpression, no inducer was used in the assays. It is worth to note, that the
expression system that we used allows simultaneous co-expression of two
genes (see above). A comparison between two different strains, BL21(DE3)
and MC4100(DE3), revealed that MC4100(DE3) cells are far more resistant
to ethidium than are BL21(DE3) cells (unpublished observation). One
explanation could be that BL21(DE3) cells are more permeable to the toxin,
due to a truncated core oligosaccharide in the cell wall (17, 18, 34-36).
Importantly, we could not observe any qualitative differences between the
strains, meaning that different EmrE-variants rendered the cells equally
resistant, providing that enough ethidium was present to kill ‘background
growth’ (=cells carrying empty plasmid vectors). Further, comparing sessile
and liquid growth of BL21(DE3) cells, we saw that the growth conditions
are important: BL21(DE3) cells growing on a plate are much more sensitive
to ethidium, compared to fellow cells growing in liquid medium. One
possible explanation is the fact that BL21(DE3) cells are not able to form
proper biofilms, and therefore sessile growth makes them more sensitive
(17). Again, we have not observed any qualitative differences between the
assays.
In the toxicity assays, we often took use of the fact that the activity of EmrE
is abolished if the conserved glutamate (E14) in the first transmembrane
helix is replaced by any other amino acid. However, as we first show in
Paper II, the glutamate can be replaced by an aspartate in one of the subunits
in a heterodimer formed by oppositely orientated EmrE-variants. Assuming
that an antiparallel dimer is a prerequisite for function, this allowed us to
42
indirectly assay the orientation of charge-altered and otherwise mutated
EmrE versions, by co-expression with EmrE variants with a known, fixed,
orientation in the membrane (generated in Paper II and coined EmrENin/Cin and EmrE-Nout/Cout, see below).
Blue-Native PAGE
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) allows for the
separation of membrane protein complexes, see e.g. (42). In Paper IV, we
solubilised radiolabelled EmrE in the mild detergent n-dodecyl-β-Dmaltoside (DDM), and analysed the presence of dimers and higher oligomers
using BN-PAGE. The oligomeric state in the micelles generally reflects the
organisation in the native membrane, however we could show that upon
heating, proteins were able to reassemble into the preferred, most stable
arrangement, regardless of starting point (Paper IV).
Cysteine labelling and crosslinking
Selective labelling and crosslinking of cysteines can be used to probe the
topology and organisation of transmembrane helices, see e.g. (109, 240). By
introducing single cysteines into a cysteine-less membrane protein, one can
determine the localization (cytoplasmic/extracytoplasmic) of the unique
residue. One can for example use membrane-impermeable 2(trimethylammonium) ethyl methanethiosulfonate (MTSET) to block any
cysteines that are exposed on the outside of the cytoplasmic membrane.
Following lysis, any unreacted cysteines can be detected by use of
MalPEG5000, a cysteine-specific reagent that causes a size shift that is
easily detected by conventional SDS-PAGE.
Cysteine crosslinking can be catalyzed by copper phenantroline. Again,
unique cysteines are introduced to the membrane protein of interest. If two
cysteines are sufficiently close, addition of copper phenantroline will induce
the formation of disulfide bridges, and dimer formation can be analyzed by
SDS-PAGE.
43
Summary of papers
Paper I
Here, we investigated the nature and occurrence of putative dual-topology
membrane proteins in bacteria. Five candidate dual-topology membrane
proteins in the cytoplasmic membrane of E. coli had in common a small size
and a weak charge bias; i.e. they have very few positively charged amino
acid residues that are evenly distributed between cytoplasmic and
periplasmic loops (57). Using C-terminal topology reporters GFP and PhoA,
we could show that in compliance with the positive-inside rule, these
proteins are topologically hypersensitive to the addition and removal of
single positively charged amino acid residues, as opposed to homologous
proteins with an unmistakable natural charge bias.
Looking for dual-topology membrane proteins in other bacteria, we found
that genes encoding putative dual-topology proteins occur as isolated
‘singleton genes’. In contrast, we also found closely spaced gene pairs
encoding homologous proteins with predicted opposite orientations in the
membrane (such as E. coli YdgE/F). Further, we came across one bacterial
membrane protein family (DUF606) that contained i) genes encoding
putative dual-topology membrane proteins, ii) paired genes encoding
homologous proteins with predicted opposite orientations in the membrane,
and iii) genes that are twice as long and that code for large membrane
proteins with homologous, oppositely orientated halves. This finding
suggested an underlying evolutionary pathway, where a gene encoding a
dual-topology membrane protein can be duplicated/fused and undergo
divergent evolution to form a family of homologous, yet topologically
mixed, proteins.
Paper II
Here we wanted to look closer at the evolutionary pathway outlined above.
We duplicated the gene encoding the dual-topology membrane protein
EmrE, and changed the duplicated genes so that they encoded proteins with
fixed, opposite, orientations in the membrane. The topologically fixed
variants were generated by the following charge manipulations: EmrENin/Cin (R29G, R82S, S107K), and EmrE-Nout/Cout (T28R, L85R,
R106A). The overall orientations of the proteins were probed using C44
terminal reporters, PhoA and GFP. Using the in vivo ethidium toxicity assay
described above, we could show that co-expression of the oppositely
orientated proteins was absolutely required for function. Also, it became
apparent that the heterodimer allows for changes that are not permitted in the
homodimer. One example is the glutamate in position 14, which can be
replaced by an aspartate in one of the subunits in the dimer, but not both.
This could be one of the reasons why paired genes encoding heterodimers
seem to be more common in nature, than single genes encoding dualtopology homodimers.
Paper III
In this paper, we wanted to look closer at the effect of positive charges on
the overall orientation of EmrE. Using mutagenesis, we systematically added
single positive charges to each of the loops of EmrE. Using the ethidium in
vivo toxicity assay described above, we could determine the topological
effect of the introduced charges. For example, we could show that EmrE
with an additional positive charge in the loop between transmembrane
helices 1-2 is only functional if it is co-expressed with EmrE-Nin/Cin,
indicating that the positive charge makes the protein insert with its N- and Ctermini on the periplasmic side of the membrane. A positive charge in the
next loop, between transmembrane helices 2-3, had the opposite topological
effect, and this particular protein was only functional if it was co-expressed
with EmrE-Nout/Cout. Importanly, we could also show that EmrE(E14D),
which of course is not functional by itself due to the replacement of E14 by
D, forms a functional dimer if it is co-expressed either with EmrE-Nin/Cin
or with EmrE-Nout/Cout, as would be expected if the wild-type protein has
dual topology.
Remarkably, most positions where we placed a positive charge had an
equally strong, and predictable, effect on the overall orientation of the
protein, from the N-terminus to the very C-terminal end. This is telling of an
exceptional topological malleability, where the whole protein remains
undecided until the very last residue has been synthesized.
Paper IV
Here we have investigated the dimeric organisation of EmrE. Although it
seems clear that monomeric EmrE can insert into the membrane in two
45
opposite orientations, the relative orientation of the subunits in the dimer has
been under debate (109). Using EmrE variants with fixed, opposite
orientations in the membrane (EmrE-Nin/Cin and EmrE-Nout/Cout), we
show that although the proteins can form parallel dimers, an antiparallel
organization of the subunits in the dimer is preferred. Cysteine crosslinking
and Blue-Native PAGE analyses of intact oligomers reveal that in
membranes, the proteins form parallel dimers only if no oppositely
orientated partner is present. Co-expression of oppositely orientated proteins
almost exclusively yields antiparallel dimers. Further, parallel dimers can be
disrupted and converted into antiparallel dimers by heating of detergent
solubilized proteins. As we have seen before, in vivo function is clearly
correlated to the presence of antiparallel dimers, and taken together our
results strongly suggest that an antiparallel arrangement of the subunits in
the dimer is more stable than a parallel organization.
46
Conclusions and perspectives
The subject of this thesis is dual-topology membrane proteins in E. coli.
Dual-topology membrane proteins can insert into their native membrane in
two opposite orientations. One prerequisite for dual topology seems to be a
weak positive charge bias, and we have shown that in compliance with the
positive-inside rule, these finely balanced proteins are very sensitive to
changes in the distribution of positively charged amino acid residues.
Further, we have shown that the dual-topology protein EmrE is topologically
undecided until the very last residue has been synthesized. This addresses
interesting questions about the insertion of these proteins. Currently, very
little is known about the mechanisms for membrane targeting and integration
of dual-topology membrane proteins. We are in the process of investigating
the SecY and/or YidC dependency of E. coli EmrE, and the future will show
whether these proteins require one, both, or neither of these insertion
machineries. Apart from the (lack of) positively charged amino acid
residues, it is not entirely clear how the dual topology of EmrE and similar
proteins is ensured. It is conceivable that the lipid composition of the native
membrane is important.
While it seems clear that monomeric EmrE is inserted in two opposite
orientations, the organisation of the subunits in the EmrE dimer is under
some debate. Our results suggest that an antiparallel organisation is a
prerequisite for function, and the preferred arrangement, however the
proteins are able to form parallel dimers if no antiparallel partner is
provided. Also, possible functional importance of higher oligomers in vivo
cannot be ruled out. It will be interesting to compare the intermolecular
arrangements of the antiparallel and parallel dimers, and investigate the
exact function of the different conformations and assemblies. This could be
done by cysteine crosslinking, mutagenesis and subsequent analyses by
Blue-Native PAGE, and computational modelling.
In nature, dual-topology membrane proteins seem to be quite rare. We
hypothesize that at least some ancestors of homologous proteins with
opposite orientations in the membrane, and of membrane proteins with
47
homologous, oppositely orientated halves, had dual topology. For EmrE, we
have shown that substitutions that abolish the function of the dual-topology
homodimer are allowed in an EmrE-derived heterodimer, providing that the
‘harmful’ substitution is present in only one of the subunits. This implies
that the heterodimer is in some sense more robust than the homodimer, and
at the same time that its evolutionary space has expanded. It is conceivable
that a series of hypothetical mutations may lead to altered specificity and an
improved function of the heterodimer that cannot occur in the homodimer
without loss of function. Our system with topologically fixed heterodimers
makes it possible to further investigate the roles of individual amino acid
residues in the two subunits of the dimer.
48
Populärvetenskaplig sammanfattning på svenska
Levande organismer består av en eller flera celler. Cellerna innehåller bland
annat arvsmassa och proteiner som katalyserar de kemiska reaktioner som
cellerna behöver för att växa, frodas och föröka sig. Alla levande celler är
omgivna av åtminstone ett cellmembran, ett oljigt hölje som håller ihop
cellen och skyddar den. I membranet sitter en stor mängd olika
membranproteiner som bildligt talat fungerar som cellernas fönster, dörrar,
antenner och gripklor: tack vare membranproteinerna kan cellerna ta upp
näringsämnen, utsöndra restprodukter och överhuvudtaget kommunicera
med sin omvärld. Många membranproteiner är väldigt specifika och släpper
bara in en typ av ämnen, t.ex. socker, medan andra skickar ut ämnen som
inte behövs längre, eller som rentav är giftiga för cellen.
Membranproteinerna sitter i membranen just så, att de kan släppa in
specifika ämnen från utsidan, och skicka ut andra ämnen från insidan.
Vi har undersökt hur membranproteiner sätts in i membranen och vad det är
som bestämmer åt vilket håll ett protein hamnar. Som alla proteiner består
även membranproteiner av aminosyrakedjor, och proteinernas struktur
bestäms av aminosyrakedjornas sammansättning. En liten grupp
membranproteiner i den vanliga E. coli-bakterien har egenheten att de kan
sättas in i cellmembranet åt två motsatta håll. Vi har visat att dessa så kallade
dualtopologi-proteiner är väldigt känsliga för ändringar i aminosyrakedjan,
särskilt med avseende på positivt laddade aminosyror. Det är väl känt att
membranproteiner ofta sitter i membranet på det sättet att positiva
laddningar hamnar inne i cellen, och genom att ändra var de positiva
laddningarna sitter i proteinet kan man bestämma åt vilket håll proteinet sätts
in i membranet.
De flesta membranproteiner sätts in i membranen samtidigt som de
syntetiseras av ribosomer. Vi har visat att dual-topologiproteiner får sin
slutgiltiga orientering i membranet först när hela proteinet har syntetiseras.
Vidare föreslår vi att genom genduplikation och evolution kan dualtopologiproteiner ge upphov till större proteiner som innehåller två motsatt
orienterade, besläktade halvor.
49
Acknowledgements
I owe thanks to so many people that I cannot possibly name all of you here,
simply because there is not enough space to do so. However I hope you
know who you are, and how much you mean to me! Tack, alla vänner!

Here are just a few ‘special thanks’ to some of you, without whom this thesis
would not exist. Först och främst vill jag såklart tacka Gunnar von Heijne den bästa handledaren man kan tänka sig. Det har, kort sagt, varit toppen!
Din schyssta inställning till forskning är klart föredömlig  Stort tack till
Mikaela Rapp för att jag fick haka på dina spännande projekt. Jag kommer aldrig - att glömma alla roliga stunder på labbet (frysen är fortfarande full av
konstrukt som har mycket sofistikerade och väl genomtänkta namn…;)). Jag
hoppas att vi får möjlighet att jobba ihop igen!  Tack Pilar Lloris-Garcerá,
Joanna SG Slusky, Daniel O Daley, Erik Granseth och Frans Bianchi för gott
samarbete. Many thanks for fruitful collaborations - it’s been great fun! Let’s
stay in touch!

Many thanks to other present and past members of the GvH/IMN-cluster:
Karin Ö, Florian, Salomé, Patricia and Nina in the ’best office’ - thanks for
nice discussions, good advice, and a lot of laughs! I wish you all the best!
Carmen, Nurzian, Rickard and now also Johannes - you all contribute to the
great atmosphere of the lab! Thank you for all good times!  Bill and Patrik,
keep up the good work! Thanks to all former office/lab mates: Marie (det var
kul att dela kontor med någon som fick en att skratta innan man ens hunnit
dricka upp sitt morgonkaffe ), Morten a.k.a. Dr Clone (särskilt tack för att
du pekade ut den gamle mossen), Roger (it was great having you in the lab…
it went a bit quiet when you left…), Carolina (jag glömmer nog aldrig våra
intressanta kulinariska erfarenheter i Bilbao…), Joy (always so kind and
helpful!), and Katrin (hör av dig om du tar båten över nån gång! ), Yoko,
Mirjam, Nadja, Tara, Andreas, Marika - many thanks to all of you for such
good times  Extra stort tack till IngMarie Nilsson - jag vet inte vad vi
50
skulle göra utan dig! Du förtjänar en stor guldstjärna!  Thank you JanWillem (for scientific input), David D (great that you’re back! Good luck!),
David V (m. familj för barnstolen som står i pentryt - den är väl använd ),
Louise, Anna, Sam, Dimitra, Susan, Mirjam - you guys in JWdG’s group
know how to throw a pub/fika - we’ve shared many good cakes  Kalle
(tack inte minst för avhandlings-mallen…), Filippa (för allt du lärt mig om
membranprotein-komplex!), Stephen, Jörg, Isolde (som tillsammans med
Ann-Louise och Erika delar plats som bästa studenten nånsin ), Rob (have
fun in the ’new’ lab! ), Johan, Mili, Minttu, and everyone else in the
’GvH/deGier/IMN/Daley/Daniels-community’. It’s been a blast! Thanks and
good luck to all of you! 
Many thanks to everyone else at DBB for making the department such an
enjoyable place! You are an inspiration.  Särskilt tack till Åke, Inger,
Elzbieta, Peter B, Pia Ä, Pia H, Mikael, Agneta, Andreas, Astrid, Lena och
alla i era grupper, inte minst för att ni tar er tid att svara på mina frågor 
Tusen tack till Stefan Nordlund för ditt enorma engagemang i
doktorandernas vardag. Tack till Ann, Maria och Lotta på sekretariatet - ni
rockar!  Tack till Håkan för att du fixar med praktiska saker och Torbjörn
och Peter N för all hjälp med e-post/datorer/skrivare. Bogos R.I.P.
Sist men inte minst: tack Martin för hjälp med molekylgrafiken, och för allt
annat också. Du och Eunike är det bästa jag vet! Puss!

51
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