Download Integration and topology of membrane proteins Carolina Boekel

Integration and topology of
membrane proteins
Carolina Boekel
Cover illustration: “Cell membrane” inspired by Mondrian
© Carolina Boekel, Stockholm 2009, pages i-49
ISBN 978-91-7155-827-5
Printed in Sweden by Universitetsservice AB, Stockholm 2009
Distributor: Department of Biochemistry and Biophysics,
Stockholm University
“If you learn a lot of little things, one day
you may end up knowing a big thing”
- B.K.S Iyengar
Contents
Abstract
i
Publications included in this thesis
ii
Introduction
4
Background
6
Biological membranes
6
Lipids
7
Structural features of membrane proteins
7
α-helical membrane proteins
8
β-barrel membrane proteins
9
Hydrophobic amino acids
9
Aromatic amino acids
9
Proline-induced turns
9
Snorkelling
10
Hydrophobicity scales
10
Biogenesis of membrane proteins in the endoplasmic reticulum
12
Targeting to the endoplasmic reticular membrane
12
The signal recognition particle
12
Post –translational translocation
13
The translocon
14
Glycosylation - Oligosaccharyl transferase
18
Signal peptidase
19
Topology - a structural model of membrane proteins
21
Membrane protein orientation
21
Single-spanning membrane proteins
22
Bioinformatics
23
Topology mapping
24
Methodology
27
Model system - Leader peptidase
27
Expression in vivo
28
Expression in S. cerevisiae
29
Results and discussion
30
Summary of papers
30
Paper I: Helix-helix interactions
30
Paper II: Opposite orientation: Nin-Cout vs. Nout-Cin transmembrane helices
32
Paper III: Insertion efficiency of the amyloid β-peptide into the ER membrane
34
Paper IV and V: Topology mapping of two eukaryotic membrane proteins
35
Acknowledgements
37
References
39
Abbreviations
aa
amino acids
Aβ
amyloid β-peptide
APP
amyloid-β precursor protein
C-terminal
carboxy-terminal
DNA
deoxyribonucleic acid
ER
endoplasmic reticulum
Lep
leader peptidase
N-terminal
amino-terminal
OST
oligosaccharyl transferase
RNC
ribosome nascent chain complex
SP
signal peptidase
SR
SRP receptor
SRP
signal recognition particle
TM
transmembrane
TMH
transmembrane helix
TMHMM
transmembrane hidden Markov model
TRAM
translocating chain-associated membrane protein
TRAP
translocon –associated protein complex
Å
Ångström
Amino acids
Ala
Alanine
Leu
Leucine
Arg
Arginine
Lys
Lysine
Asn
Asparagine
Met
Methionine
Asp
Aspartic acid
Phe
Phenylalanine
Cys
Cysteine
Pro
Proline
Gly
Glycine
Ser
Serine
Gln
Glutamine
Thr
Threonine
Glu
Glutamic acid
Trp
Tryptophan
His
Histindine
Tyr
Tyrosine
Ile
Isoleucine
Val
Valine
Prediction server
ΔG-pred
Predicition of ΔG for TM-helix insertion
http://www.cbr.su.se/DGpred/
Sfinx metaserver
http://sfinx.cgb.ki.se
Prediction of transmembrane topology
Abstract
Membrane proteins comprise around 20-30% of most proteomes. They play
important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug
design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting
channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how
the translocon recognizes the transmembrane helices and integrates them
into the membrane.
We have investigated the sequence requirements for the translocon-mediated
integration of a transmembrane α-helix into the ER by challenging the Sec61
translocon with designed polypeptide segments in an in vitro expression
system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins
that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration
efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.
Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.
Publications included in this thesis
This thesis is based on the following publications, which will be referred to
by their Roman numerals.
I. Meindl-Beinker NM, Lundin C, Nilsson I, White SH, von Heijne G.
(2006) Asn- and Asp-mediated interactions between transmembrane helices
during translocon-mediated membrane protein assembly.
EMBO Rep. 7(11): 1111–16.
II. Lundin C, Kim H, Nilsson I, White SH, von Heijne G. (2008)
The molecular code for protein insertion in the ER membrane is similar for
Nin-Cout vs. Nout-Cin transmembrane helices.
Proc. Natl. Acad. Sci. USA. 105(41):15702-07.
III. Lundin C, Johansson S, Johnson AE, Näslund J, von Heijne G, Nilsson
I. (2007) Stable insertion of Alzheimer Aβ peptide into the ER membrane
strongly correlates with its length.
FEBS Lett. 581(20):3809-13.
IV. Lundin C, Nordström R, Wagner K, Windpassinger C, Andersson H,
von Heijne G, Nilsson I. (2006) Membrane topology of the human seipin
protein.
FEBS Lett. 580(9):2281-4.
V. Lundin C, Käll L, Kreher S A, Kapp K, Sonnhammer E L, Carlson J R,
von Heijne G, Nilsson I. (2007) Membrane topology of the Drosophila
OR83b odorant receptor.
FEBS Lett. 581(29):5601-4.
Reprints were made with permission from the publishers.
Additional publications:
•
Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H,
Nilsson I, White S.H, and von Heijne G. (2005). Recognition of
transmembrane helices by the endoplasmic reticulum translocon.
Nature. 433(7024): 377-81.
•
Lerch-Bader M, Lundin C, Kim H, Nilsson I, von Heijne G. (2008)
Contribution of positively charged flanking residues to the insertion
of transmembrane helices into the endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA. 105(11):4127-32.
•
Enquist K*, Fransson M*, Boekel C* , Bengtsson I, Geiger K, Lang
L, Pettersson A, Johansson S, von Heijne G, Nilsson I. (2009)
Membrane-integration characteristics of two ABC transporters,
CFTR and P-glycoprotein. *These authors contributed equally to
this work.
J. Mol. Biol. Feb 21 (Epub ahead of print)
Introduction
All living cells contain proteins that carry out specialized functions within
the membrane or aqueous spaces. Half of all proteins in a typical cell are
transported across or into a membrane. How are proteins synthesized in the
cytoplasm of the cell and inserted across or into the membranes?
The eukaryotic cell contains both a plasma membrane and internal membranes. These internal membranes create vesicles and organelles such as the
nucleus, endoplasmic reticulum (ER), mitochondrion, chloroplasts, peroxisomes, Golgi and lysosome/vacuoles, each with a specialized function.
George Palade defined the basic structure of the secretory pathway in eukaryotic cells and in 1970, Günter Blobel performed experiments on the
translocation of proteins across membranes. He discovered that many proteins have a short amino acid sequence at one end that functions like a postal
code for the target organelle, a signal sequence, and the “signal hypothesis”
was born (Blobel and Sabatini, 1971).
In the ER, the N-terminal postal code (signal sequence) of the protein is recognized by the signal recognition particle (SRP) while the protein is still
being synthesized by the ribosome. The synthesis pauses while the ribosome-protein complex is transferred to an SRP receptor at the ER. There, the
nascent protein is inserted into a protein channel (the transclocon) that spans
the ER membrane. The translocon is also responsible for retro-translocation
of misfolded polypeptides destined for degradation in the cytosol by the ERassociated degradation (ERAD) mechanism (Nakatsukasa and Brodsky,
2008; Wiertz et al., 1996).
Proteins that are localized to the Golgi, lysosome/vacuole and plasma membrane are first inserted in the ER. Within the ER, a 14-residue oligosaccharyl
core is attached to glycoproteins. Transmembrane proteins are translocated
through the membrane by the translocon, until the process is interrupted by a
stop-transfer sequence. The amino acid chain of a transmembrane protein
can pass back and forth across the membrane one or several times.
4
This thesis is about how membrane proteins insert into the ER membrane. In
papers I-III, we have investigated the sequence requirements for translocation and insertion of artificial and natural transmembrane segments. Very
few structures of membrane proteins are known and to broaden our understanding of structure, we have performed topology mapping on two biologically interesting membrane proteins of unknown structure, paper IV-V: the
human seipin protein and the Drosophila melanogaster odorant receptor
OR83b.
5
Background
Biological membranes
Membranes define the external boundaries of cells and regulate the molecular traffic in and out of the cell. The membrane is a dynamic lipid bilayer
that is selectively permeable; it lets some compounds through, while excluding others. In order to do this, the cell membrane contains proteins embedded in the lipid bilayer that act as mediators. These proteins are referred to as
membrane proteins. All membrane proteins contain both a hydrophobic part
and a hydrophilic part that enable them to be anchored in the membrane and
at the same time be in contact with the surrounding environment.
Singer and Nicholson defined the biological membrane (Figure 1A) as a
bilayer of amphipathic lipids with proteins floating around freely as icebergs
in a sea (Singer and Nicolson, 1972). In addition, some membranes also
have carbohydrates (mono- and oligosaccharides) attached to their surface.
A
B
Figure 1. A. Schematic representation of a biological membrane of lipids, cholesterol and membrane proteins. B. Chemical structure of a membrane lipid, phospatidyletanolamin.
6
Lipids
Membrane lipids are amphipathic molecules with a hydrophilic head and a
hydrophobic tail (Figure 1B). For most lipids, the tail region is made up of
two acyl carbon chains, typically 16-18 carbons long and differing in the
degree of saturation (double bonds).
Due to thermodynamic constrains in an aqueous environment, the lipids selfassemble into a bilayer where the lipid tails point towards the interior, while
the headgroups face the hydrophilic exterior on each side of the membrane.
Cylindrical lipids promote the formation of a flat lipid bilayer. A lipid is
cylindrical when the head group has approximately the same size as the hydocarbon tail. If the headgroup is larger or smaller than the hydrocarbon
tails, the conical shape induces curvature in the lipid monolayer (Gruner,
1985).
The head groups can also vary in polarity; they can be neutral, zwitterionic
or negatively charged. Most biological membranes are made of phospholipids, glycolipids and cholesterol, a neutral lipid containing a ring structure
that breaks the tight packing of fatty acid chains, creating a more fluid membrane. Biological membranes contain many different kinds of lipids and the
lipid composition gives the different membranes their specific characteristics
such as curvature, fluidity and rigidity.
Structural features of membrane proteins
Membrane proteins have amphipathic structures that reflect the membrane in
which they reside. They have polar surfaces that interact with the aqueous
solution and with the lipid head groups, and nonpolar surfaces that interact
with the nonpolar interior of the lipid bilayer. As a result of this, they are
neither soluble in aqueous solution nor in non-polar solvents.
Membrane proteins can be classified into two categories: integral (intrinsic)
and peripheral (extrinsic), based on the type of lipid- protein interactions. An
integral membrane protein contains residues with hydrophobic side chains
that interact with the fatty acyl chains of the membrane phospholipids and
anchors the protein to the membrane in a way that it cannot be extracted by
high pH. Most integral proteins span the entire membrane layer and are
therefore called transmembrane proteins.
Transmembrane proteins can assume the structure of helix bundles or βbarrels (von Heijne, 1996) due to the stabilization by the internal hydrogen
bonds (Figure 2).
7
Figure 2. Integral membrane proteins. Ribbon diagram of a β-barrel membrane
protein (left) and an α-helical membrane protein with 7 transmembrane helices
(right). The yellow lines indicate the borders of the membrane.
Peripheral membrane proteins are associated with the membrane through
interactions with the hydrophilic domain of integral proteins and/or with the
polar head groups of membrane lipids. Carbonate extraction at high pH is
often used to extract peripheral membrane proteins from the membrane.
α-helical membrane proteins
When there are no water molecules available to form hydrogen bonds with
the carbonyl oxygen and amide nitrogen of the peptide bond, internally hydrogen bonded α-helices give the most stable conformation (Hermansson
and von Heijne, 2003). Genomic analyses of several organisms indicate that
20-30% of all open reading frames code for helix bundle membrane proteins
(Krogh et al., 2001).
The most obvious feature of the helix-bundle class proteins is a stretch of
around 26 hydrophobic amino acids that will form a transmembrane a-helix,
which is roughly perpendicular to the membrane plane (Ulmschneider et al.,
2005). Between 15 and 43 amino acids are generally considered the boundaries, in order to successfully form an α–helix that spans the membrane
(Granseth et al., 2005). When the polypeptide chain is translocated by the
translocon, the hydrophobic segments will partition into the membrane. The
favorable partitioning of the hydrophobic side chains into the membrane and
the formation of backbone hydrogen bonds overcome the unfavorable entropy of folding the backbone (Faham et al., 2004; Popot and Engelman,
1990). Both single-spanning and polytopic membrane proteins exist.
8
In prokaryotes, most α–helical membrane proteins are located in the inner
membrane, but recently a polysaccharide transporter (Wza) has been shown
to form a transmembrane helix bundle in the outer membrane of Escherichia
coli (Dong et al., 2006).
β-barrel membrane proteins
Another possibility for hydrophobic amino acid sequences to span the membrane is the β-barrel, stabilized by hydrogen bonds between antiparallel βstrands. Such proteins are found in the outer membrane of Gram-negative
bacteria (Kleinschmidt and Tamm, 1996) and are predicted to be present in
the outer membrane of mitochondria and chloroplasts (Schleiff et al., 2003).
Hydrophobic amino acids
The hydrophobic domain of a TM α–helix is mainly composed of hydrophobic amino acid residues (Ile, Leu, Val, Ala, Phe and Gly). These residues
account for 63% of the residues in TM helices (Ulmschneider and Sansom,
2001).
Alanine is a hydrophobic amino acid on the threshold between those amino
acids that promote membrane integration of membrane proteins and those
that do not. Previous work (Nilsson et al., 2003a) has shown that Ala has a
slight tendency to prefer the lipid-water interface region over the central part
of the membrane.
Aromatic amino acids
Aromatic amino acids are often located at the lipid-water interface, ±15 Å
from the center of the membrane. Trp and Tyr are enriched near the ends of
the helices, suggesting that they interact favorably with the lipid headgroups.
Phe, on the other hand is more abundant in the central core region of the TM
helices. Trp, Tyr and Phe tend to push the transmembrane helix into the
membrane when inserted in positions flanking the TM, and Trp pulls the
transmembrane helix toward the lipid-water interface when inserted inside
the TM segment (Braun and von Heijne, 1999).
Proline-induced turns
Previous work has shown that Pro breaks a 40 residue long poly–Leu TM
helix when placed in the central ten positions (Nilsson and von Heijne,
1998). The loss of hydrogen bonds and the steric problems caused by the Pro
residue makes it energetically more favorable to place Pro in a tight turn near
the membrane/water interface than to force it into the center of a long, mem9
brane-embedded TM helix (Nilsson et al., 1998) . To be effective as a turnpromoter, the Pro needs to be placed in the middle of a 30 residues long hydrophobic stretch (Monne et al., 1999). Pro has also been shown to be a
topology determinant of the integral membrane protein stomatin. Some proteins such as stomatin have a unique hairpin-loop topology, possibly created
by the helix-breaking properties of Pro (Kadurin et al., 2008).
Snorkelling
The so-called “snorkel effect” occurs when charged residues with long aliphatic side chains can stretch along the surface of the helix (Chamberlain et
al., 2004). This allows the charged terminal group to reach into the lipid
headgroup region, even when the Cα is five to six residues below the membrane/water interface.
A previous study (Monne et al., 1998) showed that the negatively charged
residues, Asp and Glu, can have a strong effect on the position of a poly-Leu
TMH in the ER membrane when they are located in the first turn of the helix. They had almost no effect when placed further into the hydrophobic
stretch.
The side chain of Asp is short and cannot “snorkel” in the same way as positively charged Arg and Lys. There may also be some repulsion between the
negatively charged residues and negatively charged phospholipids head
groups (van Klompenburg et al., 1997).
Hydrophobicity scales
The hydrophibicities of the individual amino acid side chains have been
measured experimentally in octanol, which serves as a model for the hydrocarbon core of a lipid bilayer (White and Wimley, 1999; Wimley et al.,
1996). The hydrophobicity of the amino acids is defined as the free energy
of transfer from water to a nonpolar liquid.
The apparent hydrophobicities of amino acid side chains vary a lot; depending on whether or not polar groups are present. Ionized and polar side chains
interact strongly with water and have lower solubilities in nonpolar solvents
because of the unfavorable energetics of placing polar groups in a nonpolar
environment. According to the octanol scale, transferring the backbone (a
Gly) is unfavorable by 1.25 kcal mol-1 per residue. This suggests that sidechain hydrophobicity drives the equilibrium in favor of insertion and that
there is a threshold hydrophobicity that would favor insertion into the lipid
bilayer (Liu et al., 1996).
10
A 20-residue polyalanine sequence would not be hydrophobic enough to
partition into the hydrocarbon core (ΔG=10 kcal mol-1), but replacing five of
the Ala with five Leu residues would make the insertion favorable (ΔG=1,25 kcal mol-1). Experiments on translocon-mediated TM insertion indicate
that this prediction is remarkably accurate (Hessa et al., 2005).
Figure 3. ΔG scale derived from H-segments with the individual aa placed in the
middle of a 19-residue hydrophobic stretch (Hessa et al., 2005).
Based on a large set of data from insertion of designed model segments, the
contribution to the free energy of membrane integration (ΔGaaapp ) could be
estimated for each of the 20 naturally occurring amino acids when placed in
the middle of a segment. As seen in Figure 3, Ile, Leu, Phe and Val promote
membrane insertion (ΔGaaapp < 0), Cys, Met, Ala have (ΔGaaapp ≈ 0) while the
polar and charged residues have (ΔGaaapp > 0).
11
Biogenesis of membrane proteins in the
endoplasmic reticulum
Targeting to the endoplasmic reticular membrane
Proteins that are going to be exported (translocated) across or inserted into
the ER membrane need both the action of the translating ribosome in the
cytoplasm and translocons located in the ER of eukaryotes or the plasma
membrane of bacteria. The translocon allows soluble polypeptides to cross
the membrane and hydrophobic transmembrane segments of membrane proteins to exit laterally into the lipid phase. Insertion and translocation of
membrane proteins can occur both co- and post-translationally and converge
at the Sec complex.
Proteins destined for translocation carry an amino-terminal signal sequence
that marks them for translocation, which is recognized by targeting factors
(Walter and Blobel, 1981a). The signal sequence can be divided into three
different regions: a positively charged N-terminal (n-region), a central hydrophobic core (h-region) and a slightly polar C-terminal (c-region), which
often contains amino acids with short side chains at the positions closest to
the cleavage site (von Heijne, 1983; von Heijne, 1985).
The signal recognition particle
In the event of co-translational insertion, the N-terminal part of the polypeptide emerges from the ribosome and is recognized by the signal recognition
particle (SRP). SRP is a ribonucleotide particle, existing in all three kingdoms of life (Keenan et al., 2001). The binding of SRP to the hydrophobic
signal sequences arrests protein synthesis (Walter and Blobel, 1981b), this
prevents the protein from premature folding or aggregation in the cytosol.
SRP docks onto the ribosomal component L23 located near the ribosomal
exit tunnel and binds to the hydrophobic signal sequence as it emerges from
the tunnel (Schaffitzel et al., 2006; Valent et al., 1997), forming a ribosome
– nascent chain complex (RNC). In eukaryotes SRP then binds GTP and
arrests elongation. The RNC-complex binds to the membrane bound SRP
receptor (SR), another GTPase, which is associated with the translocon. The
binding of SRP to SR activates the GTPase activities of both SRP and SR,
whereupon the SRP disengages from the SR and the ribosome, the protein is
transferred to the translocon, and elongation of the protein continues (Figure
4) (White and von Heijne, 2004).
12
Figure 4. Co-translational translocation. The model is based on the eukaryotic system, but is probably similar for all organisms (Rapoport, 2007).
The mammalian SRP consists of a 7S RNA and six proteins (SRP9, 14, 19,
54, 68, 72) and it has two domains, the Alu domain (SRP14-SRP9 and 7SL
RNA) and the S domain (forked region of 7SL RNA and the remaining four
proteins) (Nagai et al., 2003). The Alu domain confers peptide elongation
arrest activity. The S domain mediates signal sequence binding and SR
docking (Halic and Beckmann, 2005).
A cryo-EM structure of a mammalian SRP bound to an active 80S ribosome
(Halic et al., 2004; Wild et al., 2004) shows how the S domain of SRP interacts with the large ribosomal subunit at the nascent chain exit site and binds
the signal sequence, while the Alu domain reaches into the elongationfactor-binding site of the ribosome, thereby causing the elongation arrest
activity.
Post –translational translocation
Post-translational translocation operates by different mechanisms in eukaryotes and bacteria. In yeast ER, the Sec61p complex together with the
Sec62p, Sec63p, Sec71p and Sec72p subcomplexes are responsible for posttranslational translocation. Mammalian homologues to Sec61p (Sec62) and
Sec63p (Sec63) have been identified and proposed to function in posttranslational translocation. However, no efficient post-translational translocation has been observed in mammalian ER (Meyer et al., 2000).
13
Eukaryotes need BiP (Kar2p in yeast), an Hsp70 homologue in the ER lumen, which utilises ATP hydrolysis to pull the polypeptide chain in a
ratchet-like manner (Matlack et al., 1999; Misselwitz et al., 1999). Bacteria
push proteins through the translocon channel by using a motor protein, the
ATPase SecA (Brundage et al., 1990; Hartl et al., 1990). In addition, the
reaction requires a cytosolic component called SecB to aid targeting to the
membrane and to prevent premature protein folding and aggregation (de
Keyzer et al., 2002; Hartl et al., 1990; Kusters et al., 1989; Weiss et al.,
1988).
The translocon
In 1975, the hypothesis that secretory proteins are translocated through the
ER membrane via an aqueous channel was first presented (Blobel and Dobberstein, 1975). Another popular theory at the time was that the signal sequence directs the spontaneous insertion of the polypeptide into the membrane, while the rest of the polypeptide is translocated through the hydrophobic core of the membrane without any assistance of a channel or proteins
(Engelman and Steitz, 1981). The mechanism of protein insertion remained
unresolved and was debated for many years due to the lack of experimental
data to support either theory. It was not until the beginning of the 1990s that
data were presented which supported the involvement of a proteinaceous
channel in protein insertion (Simon and Blobel, 1991).
The translocon components that form the protein-conducting channnel in the
ER membrane were first identified by photocrosslinking (Krieg et al., 1989).
Today we know from X-ray crystallography that Sec61 is a heterotrimeric
complex (Van den Berg et al., 2004). It consists of α-, β, and γ- subunits
(Sec61p, Sbh1p and Sss1p in yeast (Table 1) (Finke et al., 1996)/SecY, G
and E in bacteria). All genomes sequenced to date encode at least one homolog of each Sec61αβγ/SecYEG subunits, suggesting that these proteins
play a universal role (Cao and Saier, 2003).
The high resolution X-ray structure of an archeal translocon (Van den Berg
et al., 2004) suggests that one copy of the Sec61 heterotrimer serves as a
functional translocation channel (Figure 5). The α-subunit is the largest with
10 TM helices, forms the central channel, and acts as the main ribosome
receptor (Kalies et al., 1994). The α-subunit can be divided into two halves
(TM1-5 and 6-10), joined at the back of the molecule by an external loop
and the single Sec61γ-subunit. A cavity with the diameter of 20-25 Å is
formed at the cytoplasmic side of the α-subunit. The tunnel through the
channel has a diameter of 5-8 Å in the middle and contains a ring of hydrophobic residues. This ring may form a seal around the translocating polypeptide, hindering the leakage of other molecules.
14
Figure 5. The structure of the Sec-translocon from Methanococcus jannaschii (Van
den Berg et al., 2004). Top view of the translocon (left) showing the α-subunit consisting of two halves, TM1-5 and TM6-10, which can open laterally between TM7-8
and 2b-3. The purple cylinder shows the possible position of a nascent chain TM
helix that is about to move into the membrane. The channel is closed from the periplasmic side by the green “plug helix”; this plug is thought to move out of the way
when the ribosome binds to the translocon.
The Sec61β- and γ-subunits span the membrane once (Esnault et al., 1994;
Van den Berg et al., 2004). The function of the Sec61β-subunit, a 10 kD Ctail-anchored transmembrane protein, is still under investigation. An interesting finding is that Sec61β (SecG in prokaryotes) is the only translocon
subunit that is not essential for cell viability in yeast and bacteria (Boisrame
et al., 1996; Finke et al., 1996; Nishiyama et al., 1994; Toikkanen et al.,
1996). Mammalian Sec61β is not required for protein translocation in vitro,
but has been shown to have an kinetic effect on co-translational translocation
(Kalies et al., 1998). In Saccharomyces cerevisiae, Sbh1p (Sec61β) is believed to act as the guanine nucleotide exchange factor for the signal recognition particle receptor (SR) (Helmers et al., 2003).
The translocon structure also shows a plug that blocks the pore and presumably closes the channel (Figure 5). Plug displacement will then open the
channel and make translocation possible. The structure also suggests that a
TM segment in nascent polypeptides can exit the translocon channel laterally
between TM2b and TM7 of Sec61. How the opening and closing of this gate
is regulated is not known. It seems that when a hydrophobic sequence appears inside the ribosome, it induces a conformational change that would be
transmitted to the lumenal side of the channel, causing the channel to close
by the binding of the protein Bip (Hamman et al., 1998). Another conforma15
tional change would then reopen ribosome-channel junction if the polypeptide chain is further elongated (Rapoport et al., 2004).
There are several lower-resolution electron-microscopy structures of ribosome-bound bacterial and mammalian translocons (Mitra et al., 2005;
Morgan et al., 2002) proposing different models for the structure. In one
model, the active translocon is proposed to be a single Sec61 complex with
the nascent polypeptide chain translocating through a narrow channel. In
another model, Sec61 is suggested to be a front-to-front Sec61 dimer with
two lateral gates partly open and facing each other, creating a large central
channel. A more recent structure shows that a nontranslating ribosome binds
a single copy of the SecY complex with the pore of SecY located under the
ribosomal tunnel exit (Menetret et al., 2007). Consequently, this copy of
SecY could form the channel. Non-translating 80S ribosomes have been
shown to associate with three to four copies of trimeric Sec61-complexes,
whereas a translating ribosome binds only a single Sec61-complex in the ER
(Kalies et al., 2008).
In mammals, additional membrane proteins are present in the vicinity of the
translocon complex. These include the signal peptidase (SP), the translocating chain associated membrane protein (TRAM), the translocon-associated
protein complex (TRAP) (Table 1) and the oligosaccharyl transferase (OST)
(Johnson and van Waes, 1999; Osborne et al., 2005).
16
Table 1. Targeting and translocon components in the ER membranes of mammalian,
S. cerevisiae and E. coli
Mammals
S.cerevisiae
E.coli
Function
SRP
SRP
SRP
signal recognition particle
Sec61α
Sec61p
Ssh1p
SecY
translocon pore component
Sec61β
Sbh1p
Sbh2p
SecG
translocon pore component
Sec61γ
Sss1p
Sss1p
SecE
translocon pore component
SecA
cytoplasmic/membrane
Primary components
associated ATPase
Sec62
Sec62p
component for posttranslational translocation
Sec63
Sec63p
component for posttranslational translocation
Sec71p
component for posttranslational translocation
Sec72p
component for posttranslational translocation
Accessory components
TRAM
YidC
involved in lateral transfer
of TMs
TRAP
involved in translocating
proteins that have prolonged access to the cytoplasm (Fons et al., 2003)
Oligosaccharyltransferase
OST
glycosylates proteins in the
(OST)
ER lumen, close to translocon pore
Bip
Kar2p
Signal peptidase (SP)
SP
Calnexin
Calnexin
lumenal Hsp70 family
member
Lep
signal peptidase
chaperone activity: binds to
glycoproteins
Calreticulin
chaperone activity: binds to
glycoproteins
17
Glycosylation - Oligosaccharyl transferase
N-linked glycosylation occurs in all eukaryotes and in a few prokaryotes
(Kowarik et al., 2006). In eukaryotes, the process is catalyzed by a large
(∼270 kDa) (Li et al., 2008) transmembrane protein complex called oligosaccharyl transferase (OST).
Many secretory and membrane proteins are N-glycosylated by the OST
complex during their translocation across the ER membrane (Figure 6)
(Chavan et al., 2005). During co-translational glycosylation, OST is in contact with both the translocon and the ribosome (Menetret et al., 2005; Wang
and Dobberstein, 1999). When a polypeptide emerges from the translocon
the oligosaccharide is added at the lumenal side, while the rest of the polypeptide is still being synthesized by the ribosome in the cytosol (Menetret et
al., 2005; Mothes et al., 1994; Whitley et al., 1996).
Figure 6. OST complex and ribosome-translocon complex with translocating nascent chain.
18
For N-linked glycosylation to occur, OST must cleave the large oligosaccharide group from dolicholpyrophosphate, transfer it to the substrate polypeptide, and catalyze the formation of a covalent bond between the oligosaccharide and the Asn in the N-X-S/T acceptor site.
The composition of the OST complex still remains under investigation. At
least nine nonidentical subunits build the multimeric mammalian and S. cerevisiae OST complex. The mammalian OST complex consist of ribophorin I
(Ost1p in S. cerevisiae), ribophorin II (Swp1p), OST48 (Wbp1p), STT3A
and STT3B (Stt3), N33 and IAP (Ost3p and Ost6p), and Dad1 (Ost2p)
(Kelleher and Gilmore, 1997; Kelleher et al., 2003; Shibatani et al., 2005). In
addition, two other putative mammalian OST subunits, DC2 and keratinocyte-associated protein 2 (KCP2), have been identified but their function still
remains to be determined (Shibatani et al., 2005).
Several laboratories have shown that the STT3 subunit is the catalytic center
(Hua et al., 2008; Nilsson et al., 2003b; Yan and Lennarz, 2002). Two STT3
isoforms exist in mammals, STT3A and STT3B. They share 70% sequence
similarity, yet they seem to have different functionality (Kelleher et al.,
2003; Wilson and High, 2007).
Ribophorin I is thought to be transiently associated with newly synthesized
membrane proteins after their departure from the Sec61 translocon. It is
thought to retain potential substrates close to the catalytic subunit of the
OST, thereby improving the N-glycosylation efficiency (Wilson et al., 2005;
Wilson et al., 2008).
Signal peptidase
Proteins that are exported to the cell surface are often synthesized with an Nterminal, cleavable signal sequence. The signal sequences are cleaved by a
type I signal peptidase (SPase) that has its catalytic domain on the lumenal
(periplasmic) side of the membrane and is anchored to the membrane by one
or two transmembrane segments located in the N-terminal part of the protein.
SPases have been identified and characterized in Gram-negative and Grampositive bacteria, mitochondria, and the ER (Paetzel et al., 2002; Tjalsma et
al., 2000). Except for the SPase found in the ER, the SPases are serine-lysine
proteases (Karla et al., 2005). The active site serine residue, Ser-90 in E. coli
SPase, is conserved in the homologous subunit within the ER SPase complex. However, the latter differs from the bacterial and organellar SPases by
having a His in place of the catalytic Lys. The SPase in the ER may use either a serine-histidine dyad mechanism or the serine-histidine-aspartic acid
triad mechanism (VanValkenburgh et al., 1999).
19
The SPase and OST complexes appear to be in close contact with the translocon because each acts on a nascent chain as it is being translocated, SPase
to cleave off the signal sequence and OST to glycosylate the polypeptide.
Rough ER microsomes contain approximately equal numbers of ribosomes,
SPase and ribophorin I. It is therefore likely that one OST and one SPase are
associated with each translocon (Johnson and van Waes, 1999).
20
Topology - a structural model of membrane
proteins
Membrane protein orientation
To get a better understanding of the structure/function relationships that define the activity of a membrane protein, it is necessary to know its membrane
topology, i.e. the number of TM segments and their location relative to the
ER membrane. Bioinformatic methods are often used for topology prediction
(Melen et al., 2003; Möller et al., 2001; Nilsson et al., 2000b). If the location
of one terminus is known, the best programs correctly predict the topology
of 65–70% of the membrane proteins with known crystal structure (Melen et
al., 2003). The topology prediction reliability is increased if the predictions
come from several different programs that agree (Melen et al., 2003; Nilsson
et al., 2000b).
What are the determining factors for how the translocon orients a signal sequence or a transmembrane segment relative to the membrane? Membrane
proteins with cleavable signal sequences are always oriented with their mature N-terminus on the luminal side, whereas two different orientations, Nin
or Nout, are possible for membrane proteins with uncleaved signal sequences.
At least three factors determine the orientation of the TMH. First, the distribution of Arg and Lys between the segments flanking the TMH: the more
positively charged segments stay in the cytosol. This is the “positive inside”
rule (von Heijne, 1989). Genome wide studies have shown that this charge
bias is present in almost all living organisms (Nilsson et al., 2005; Wallin
and von Heijne, 1998). However, it is not an absolute truth. Cytoplasmic
domains with a net negative charge have been found (Allard and Bertrand,
1992), and there are examples of when the “positive inside” rule can be
overridden. This can happen when negatively charged residues are present in
high numbers (Nilsson and von Heijne, 1990), when negative charges lie
within a window of six residues from the end of a highly hydrophobic TMH
(Rutz et al., 1999), or when negative charges flank a marginally hydrophobic
TMH (Delgado-Partin and Dalbey, 1998). The molecular mechanism behind
the “positive inside” rule and the apparent dominance of positively over
negatively charged residues remains to be fully understood.
The second factor is the folding characteristics of the N-terminal domain
prior to the TMH: only regions without stable tertiary structure can be translocated (Denzer et al., 1995). The third factor is the length of the hydrophobic sequence: longer sequences favor localization of the N terminus to the
ER lumen (Sakaguchi et al., 1992; Wahlberg and Spiess, 1997). In addition,
the orientation of the first TM in some multispanning membrane protein
21
decides the orientation for the following TMHs (Wessels and Spiess, 1988).
There are also cases where the internal TMHs have a preferred orientation
regardless of the preceding TMHs (Gafvelin and von Heijne, 1994; Locker
et al., 1992; McGovern et al., 1991; Nilsson et al., 2000a; Sato et al., 1998).
The lipid composition of the bilayer also has influence on the final topological organization. A recent study on E. coli lactose permease (LacY)
(Bogdanov et al., 2008) shows that phosphatidylethanolamine (PE) restrains
the translocation potential of negative residues in favor of the cytoplasmic
retention potential of positive residues, giving an explanation to the dominance of positive over negative amino acids as co- or post-translational topological determinants.
Single-spanning membrane proteins
Single spanning membrane proteins can be classified according to their orientation in the membrane (Figure 7). Type I membrane proteins are targeted
to the ER membrane by a cleavable N-terminal signal sequence (Blobel,
1983; von Heijne, 1990). Once in the membrane, they are anchored in the
membrane with a stop-transfer (ST) sequence and oriented with an exoplasmic N-terminus and the C-terminus in the cytosol.
Membrane proteins of type II have a cytoplasmic N-teminus and a exoplasmic C-terminus. A signal anchor composed of 18-25 mainly apolar residues
anchors the protein in the membrane and induces the translocation of the Cterminal end across the membrane.
Type III membrane proteins are inserted into the membrane with an
exoplasmic N- and a cytoplasmic C-terminus. This type of proteins is anchored in the membrane by a reverse signal anchor-sequence that induces the
translocation of the N-terminal end across the membrane. Both the signal
anchor and the reverse signal anchor are recognized and targeted to the
membrane by the SRP.
22
Exoplasm
Cytoplasm
I
I
II
I
III
Figure 7. Schematic illustration of type I, II and III membrane proteins.
Bioinformatics
Determining the topology of a membrane protein however, is a greater challenge than determining its amino acid sequence. Thanks to the fast development of computer technology there are now advanced prediction methods for
membrane proteins. It is possible to predict weather a protein is a α-helical
membrane protein, its topology, if it contains a signal sequence and its organelle localization. The only requirement is that the amino acid sequence is
known.
There are many different prediction algorithms in use today. The first prediction methods simply evaluated the hydrophobicity of individual residues;
regions with several hydrophobic residues were predicted to be TM domains
(Kyte and Doolittle, 1982). The dense alignment surface (DAS) method analyzes the frequency with which groups of amino acids are found in the TM
domains of proteins in the test set (Cserzö et al., 1997). The latest generation
of topology-prediction programs uses machine-learning algorithms called
hidden Markov models (HMM), which are trained by analyzing the residues
that tend to occupy defined regions in integral membrane proteins. Two such
algorithms, TMHMM and HMMTOP, assess five or seven (respectively)
defined regions of an integral membrane protein, such as the helix core, the
TM domain boundaries and cytosolic and luminal parts. Instead of looking at
the probability of individual or groups of amino acids to populate each region as in TMHMM, HMMTOP assigns topology by comparing the residues
found in one region with those found in other regions (Sonnhammer et al.,
1998; Tusnady and Simon, 1998). To evaluate a protein, the programs look
for distribution of amino acids in patterns similar to those defined in the
training set.
Membrane protein topology prediction programs generally give four different kinds of information: (1) whether or not the protein is likely to be an
integral membrane protein; (2) how many TMHs the protein has; (3) the ori23
entations of each of the TMHs; and (4) the boundaries of the membrane and
non-membrane domains (Ott and Lingappa, 2002). No program is perfect,
and incorrect predictions can appear from several different sources. The hydrophobic core of a soluble protein can be misidentified as a TM domain and
the program can miss short TM domains or TM domains containing charged
residues.
Topology mapping
Multispanning membrane proteins cross the membrane in a zigzag way and
expose their hydrophilic loops alternately in the different compartments that
are separated by the membrane. To determine the protein topology or verify
predicted topology models, the TM domains must be confirmed and the hydrophilic loops must be localized to one compartment (van Geest and
Lolkema, 2000).
PhoA
One of the first proteins to be used as a topology reporter is PhoA, which has
been widely used for identifying periplasmic loops of membrane proteins in
E.coli (Manoil et al., 1990). Two disulfide bonds have to forme in order fore
PhoA to become catalytically active. Once active it catalyses the hydrolysis
of phosphate groups located in the periplasm of bacteria. The disulfide bonds
can only form when PhoA is located in the periplasm; if the protein is located in the cytoplasm PhoA will not fold correctly and remains inactive.
PhoA is resistant to proteases when located in the periplasm, but not when
located in the cytoplasm (Roberts and Chlebowski, 1984). Both of these
features can be used to map topology.
GFP
Green fluorescent protein (GFP) is a (26.9 kDa) protein that originally
comes from the jellyfish Aequorea victoria (Shimomura et al., 1962). GFP is
widely used as a cytoplasmic reporter as it can be fused to the C-terminal
end of the protein of interest (Drew et al., 2002; Rapp et al., 2004). GFP
must fold correctly in order to become fluorescent and it can only fold correctly when it is located in the cytoplasm of E.coli (Feilmeier et al., 2000).
Glycosylation mapping
An example of a target site that can be used for topology mapping is the Nglycosylation site that can be introduced at specific positions in the protein
by site-directed mutagenesis. N-linked glycosylation can only occur in the
ER lumen, due to the location of the active site of OST. Addition of the oligosaccharide to a target site in a protein results in an increase of the molecular mass by ∼2 kDa compared to the unglycosylated form and can be detected by SDS-PAGE (Chavez and Hall, 1991).
24
In the glycosylation mapping approach, the lumenally oriented active site of
the OST is used as a fixed point of reference against which the position of a
TMH in the ER membrane is measured. Point mutations in a TMH that affect the position in the membrane will change the minimal glycosylation
distance (Nilsson and von Heijne, 2000). The minimal glycosylation distance
(MGD) is the number of residues in the nascent chain needed to bridge the
distance between a given reference residue at the luminal end of the TMH
and the OST active site (Monne et al., 1998). For a typical TMH, the MGD
is about 10 residues (Figure 8).
Figure 8. Determination of the "minimal glycosylation distance" (MGD). The MGD
is defined as the minimum number of residues required between the lumenal end of
a transmembrane segment (white) and the active site of oligosaccharyl transferase
(Y) (Mingarro et al., 2000).
Protease-protection assay
The proteases trypsin and proteinase K can be added to spheroplasts or microsomes in order to confirm the topology of a membrane protein. These
enzymes degrade all exposed domains, while unexposed parts localized in
the bacterial cytoplasm or ER lumen remain undegraded. The proteaseprotected part can then be detected by SDS-PAGE.
Epitope tags
Epitope tagging is a recombinant DNA method by which a protein encoded
by a cloned gene is made immunoreactive to a known antibody. Protein detection is then performed by highly specific anti-tag antibodies, eliminating
the need for protein-specific antibodies. Commonly used epitope tags include glutathione-S-transferase (GST), cMyc, hexahistidine (6X-His),
FLAG, maltose binding protein (MBP), β-galactosidase, GAL4 and GFP
(Patton, 2002).
25
In yeast the HA-tag (hemagglutinin influenza virus epitope tag (Kolodziej
and Young, 1991)) is commonly used as an epitope tag. The HA-tag is a
small segment of the viral hemagglutinin coat protein that can be incorporated in gene expression vectors as an epitope tag. Hemagglutinin antibodies
then recognize the HA-tag, and the expressed protein can then be determined
by Western blotting.
26
Methodology
Model system - Leader peptidase
Leader peptidase (Lep) is a 233 amino acid long protease located in the inner
membrane of E. coli, where its function is to remove signal peptides from
secretory proteins. It is anchored to the membrane by two N-terminal transmembrane segments, H1 and H2, and has a small polar cytoplasmic domain
(P1) and a large C-terminal periplasmic domain (P2) (Wolfe et al., 1983).
The H1 segment is more hydrophobic than H2 and can insert into the lipid
bilayer in the absence of H2. H2 on the other hand needs the aid of H1 for
proper membrane integration (Heinrich and Rapoport, 2003). The X-ray
crystal structure of the soluble P2 domain and site-directed mutagenesis
studies have shown that Ser 90 and Lys 145 in the P2 domain are essential
for the catalytic activity (Paetzel et al., 1998; Sung and Dalbey, 1992;
Tschantz et al., 1993).
Figure 9. Topology of wild-type leader peptidase.
27
Lep inserts co-translationally into microsomes when translated in vitro and
adopts its native topology with the P1 loop facing the cytoplasm and both the
N and C-termini in the ER lumen (Nilsson and von Heijne, 1993). Lep can
be glycosylated in a eukaryotic in vitro transcription/translation system supplemented with dog pancreas microsomes. A single asparagine (N)-linked
glycosylation site (Asn215-Glu-Thr) is present in the large periplasmic domain (P2) of Lep (wt).
Expression in vivo
There are many expression vectors and host cell systems available for eukaryotic protein expression. Bacterial expression systems, especially those
based on E. coli vectors, are often used. However, these have turned out to
be less suitable for overexpression of mammalian membrane proteins. The
main reason for this is that many post-translational modifications and processing events that are required for correct folding of mammalian proteins, do
not exist in prokaryotic cells. Additionally, insertion of recombinantly expressed mammalian membrane proteins into bacterial membranes may cause
severe toxicity to the host cells, or induce the formation of inclusion bodies.
Semliki Forest virus (SFV) vectors have been applied for the expression of
recombinant integral membrane proteins in a wide range of mammalian host
cells (Lundstrom, 2003). The SFV expression system is based on the expression vector pSFV1, derived from the Semliki Forest virus, which is an enveloped single-stranded RNA virus. The vector encodes the single stranded
RNA genome of a recombinant form of the virus in one operon, into which
the target gene is cloned. The recombinant virus is unable to form viral particles due to the absence of three structural proteins necessary for particle
formation (Liljeström and Garoff, 1991). If the recombinantly produced
RNA is synthesized in vitro and subsequently transfected into cells, it will
drive a self-replication process and its own capping, leading to massive production of viral and recombinant proteins, while competing out the host protein synthesis.
28
Expression in S. cerevisiae
S. cerevisiae is another expression system for eukaryotic proteins that has
many of the same capabilities as mammalian cells to secrete and modify
proteins.
An efficient transformation system for S. cerevisiae is based on homologous
recombination (Oldenburg et al., 1997). In vivo recombination takes place
between the vector and a restriction fragment whose ends bear homology to
plasmid sequences, removing the need for an in vitro ligation reaction (Ma et
al., 1987).
29
Results and discussion
Summary of papers
The major aim of this thesis has been to define the sequence requirements
for insertion of membrane proteins into the ER membrane. We have also
performed topology mapping of two biologically interesting membrane proteins with previously unknown topology, the human seipin protein and the
Drosophila odorant receptor OR83b.
Paper I: Helix-helix interactions
We wanted to investigate whether and to what extent inter-helix hydrogen
bonding can drive the process of translocon-mediated TM helix insertion
itself, and if the separation between the two helices within the sequence may
influence these interactions.
It is known that hydrogen bonding between successive TM helices can have
a favorable effect on TM helix interactions in detergent micelles or model
lipid membranes. A single Asn or Asp in a TM helix provides a strong driving force for dimer or trimer formation (Choma et al., 2000; Gratkowski et
al., 2001; Zhou et al., 2000). Because dimerization occurs in a region of low
dielectric constant, it is likely that the carboxyl-containing side chain of Asp
is protonated. It has long been known that carboxylic acids form strong hydrogen bonds, especially in a low dielectric solvent (Pauling and Brockway,
1934; Pauling and Sherman, 1934) .
30
Figure 10. To measure the effect of Asp- or Asn-mediated interactions between
H2´and H segments, two constructs were compared for each H segment: one with a
19-Leu H2´segment (I) and one with an H2´segment in which one or two of the Leu
residues have been replaced by Asp or Asn residues (II).
By analyzing model protein constructs in which zero, one, or two Asn or
Asp residues were placed in two neighboring hydrophobic segments (Figure
10), it was found that ΔGapp (contribution to the free energy of membrane
integration of a marginally hydrophobic H-segment) was significantly reduced only if both the H2’-segment and the H-segment contained two Asn or
Asp- residues spaced one helical turn apart in both H2’ and H-segments. We
conclude that inter-helix interactions, which are important in natural proteins
(Heinrich and Rapoport, 2003; Mitra et al., 2005), can be treated as a second-order correction in the calculation of ΔGapp values based on the biological hydrophobicity scale (Hessa et al., 2005).
31
Paper II: Opposite orientation: Nin-Cout vs. Nout-Cin
transmembrane helices
Transmembrane a-helices in integral membrane proteins can have two orientations in the membrane: Nin-Cout or Nout-Cin. Previous studies of model NoutCin transmembrane segments have led to a detailed, quantitative picture of
the “molecular code” that relates amino acids sequence to membraneinsertion efficiency in vivo (Hessa et al., 2007). However, if the same code
also applies to Nin-Cout transmembrane helices was not known. In this project, we found that the contributions of individual amino acids to the overall
efficiency of membrane insertion are similar for the two kinds of helices, and
that the threshold hydrophobicity for membrane insertion can be up to ~1
kcal/mol lower for Nin-Cout compared to Nout-Cin transmembrane helices,
depending on the neighboring helices.
The mechanisms of translocon-mediated recognition of Nout-Cin and Nin-Cout
TM helices thus seem to be very similar, even though Nin-Cout TM helices
must re-initiate translocation of the downstream part of the protein while
Nout-Cin TM helices enter the translocon channel as part of a translocating
nascent chain (Figure 11).
Figure 11. Membrane insertion of Nout-Cin and Nin-Cout transmembrane helices. (A)
Nout-Cin TM helices enter the translocon channel as part of a translocating
nascent chain. (B) The entry of the H-segment into the translocon triggers
translocation of the downstream part of the nascent chain. The H-segment
integrates into the membrane with an Nin-Cout orientation.
32
We also found that when the TM helix serves as an ER-targeting signal it
has to interact with SRP before encountering the translocon, consequently
the threshold hydrophobicity is markedly higher. This suggests that many
TM helices in multi-spanning membrane proteins will not by themselves be
able to trigger SRP-mediated targeting to the ER translocon.
33
Paper III: Insertion efficiency of the amyloid βpeptide into the ER membrane
In this study we wanted to investigate the possible retention in the ER membrane of different amyloid β-peptides (Aβ) from the amyloid precursor protein (APP). We chose different Aβ-fragments corresponding to those known
to be produced in vivo from APP due to processing by β- γ- ε- and ζsecretases.
Figure 12. Model for Aβ-fragment integration. Mature APP is cleaved by βsecretases, releasing the soluble N-terminal fragment (APPsβ). The C-terminal
fragment (CTF) is produced by the ε-secretase cleavage together with the Aβ 48-49
membrane-bound fragments. Longer Aβ fragments are futher processed by ζ/γsecreatses, leaving Aβ 46 partly in the membrane while shorter fragments (Aβ 4045) are realeased into the ER lumen.
Using in vitro translation in the presence of microsomes, we demonstrated
that different Aβ segments are integrated into the ER membrane to different
degrees, depending on their overall length, hydrophobicity and helix potential (Figure 12). This might also affect their access to the secretases in the
ER membrane and the ability of Aβ to form toxic aggregates.
34
Paper IV and V: Topology mapping of two
eukaryotic membrane proteins
Using the glycosylation mapping technique both in vitro and in baby hamster
kidney (BHK) cells, we determined the topology of two membrane proteins
with unknown topology, the human seipin protein and the Drosophila odorant receptor OR83b. We first determined the topology by using multiple
prediction programs.
The seipin protein was predicted to be a transmembrane protein with two
transmembrane segments between residues 95–117 and residues 294–316
respectively. Insect ORs lack homology to G-protein-coupled receptors in
vertebrates (Wistrand et al., 2006) and were predicted to be transmembrane
proteins with seven transmembrane segments and an intracellular Nterminus and extracellular C-terminus. These predictions were then tested by
in vitro transcription/translation of a series of variants with glycosylation
sites added to different parts of the proteins.
A
B
Figure 13. Topology models for OR83b (A) and seipin (B).
Our results suggest a Ncyt-Ccyt topology for the 462 residue long seipin protein and a Ncyt- Cexo topology for the OR83b (Figure 13). Our result have also
been confirmed by later studies, showing that seipin is an ER membraneresident protein with a luminal loop and with both N- and C- termini facing
the cytoplasm (Ito et al., 2008).
Other studies on the OR83b placed its N-terminus in the cytoplasm (Benton
et al., 2006) and showed that OR83b is a conserved member of the insect OR
family and heterodimerises with other ORs, forming receptor complexes.
35
Recent work suggests that heteromeric insect ORs comprise a new class of
ligand-activated non-selective cation channels with no homology to any
previously described ion channel (Sato et al., 2008).
36
Acknowledgements
Tack till alla som förgyllt min doktorandtid, ett särskilt STORT TACK till:
Gunnar, min handledare, för din oändliga kunskap om membranproteiner
och din entusiasm inför alla resultat, bra som dåliga. Tack för all uppmuntran och frihet du har gett mig genom åren.
IngMarie, min andra handledare, för din kunskap, arbetsamhet och hjälpsamhet.
Joy, the “yeast guru”, for your kindness, for reading my thesis and giving me
the opportunity to work with you.
Stefan, för ditt stöd, uppmuntran och att du alltid är rättvis.
Agneta, för att jag alltid får nya utmaningar i undervisnings värld.
Peter Brzezinski och Birgitta Norling för att ni varit mina engagerade utvärderare.
My coauthers, thank you for great collaborations! Arthur Johnson, Christian
Windpassinger, Erik Sonnhammer, Helena Andersson, Jan Näslund, John
Carlson, Katja Kapp, Klaus Wagner, Lukas Käll, Mirjam Lerch, Nadia Meindl-Beinker, Rickard Nordström, Scott Kreher, Stephen White.
DBB/CBR people: Filippa, för alla fikastunder och diskutioner om livet i
största allmänhet. Tack för all hjälp med både undervisning och träningstips.
Susanna, för din entusiasm för forskning! Tack för allt kul vi hade i Bilbao
och för att du sprider lite kultur till DBB. Kalle S, för alla trevliga luncher
och fikastunder. Marie för att du alltid får oss att skratta. My room-mates,
Joanna, Morten, Pilar for your help, generosity and being great collegues!
Sofia, för en rolig tid i Cambridge och all hjälp med Alzheimer pappret!
Karin, for your kindness and patience in the lab. Karl E, för att du var en så
bra student och kollega.
Jan-Willem, for importing Jorrit, dankjewel! For stealing pepernoten and
always being enthusiastic about science.
37
Past and present members of the GvH cluster: Isolde, Salomè, Roger, Tara,
Marika, Mikaela, Yoko, Dan, David W, Sam, Louise, Mirjam K, Susan,
David D, thank you for your support and knowledge. Keep up the good
work!
Everyone at CBR and DBB corridors, thank you!
Mina lärare på Yogashala Stockholm, som lär mig både om yoga och livet!
Danshögskolan: Anna Karin, Marre, Kicki, Anette, Monica för den fantastiska tiden på Confidencen!
Mina vänner som stått ut med mig under avhandlingsskrivandet! Särskilt
tack till: Karolin och Alex, för alla roliga stunder på molekylärbiologlinjen,
Tovetorp, möhippor och bröllop! Minna, för att du har orkat lyssna på mina
doktorandproblem genom åren och alltid är en fantastisk vän!
My dear family: Fam. Boekel, Christa, Richard, Sietse, Arne for your generosity and love. Pappa och Solveig för alla middagar och härliga dagar i Dalarna.
Mamma, för din omsorg och ditt stöd. Tack för alla miljoner gånger du ställt
upp för mig och Jorrit och för all uppmuntran.
Jorrit, för att du får mig att skratta. För all kärlek, glädje och lycka!
Du är bäst!
38
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