Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Membrane-protein integration and the
role of the translocation channel
Tom A. Rapoport1, Veit Goder1, Sven U. Heinrich1 and Kent E.S. Matlack2
1
Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston,
MA 02115, USA
2
Virtual Text, 1100 Massachusetts Avenue, Cambridge, MA 02138, USA
Most eukaryotic membrane proteins are integrated into
the lipid bilayer during their synthesis at the endoplasmic
reticulum (ER). Their integration occurs with the help of a
protein-conducting channel formed by the heterotrimeric
Sec61 membrane-protein complex. The crystal structure
of an archaeal homolog of the complex suggests
mechanisms that enable the channel to open across
the membrane and to release laterally hydrophobic
transmembrane segments of nascent membrane proteins into lipid. Many aspects of membrane-protein
integration remain controversial and poorly understood,
but new structural data provide testable hypotheses.
We propose a model of how the channel recognizes
transmembrane segments, orients them properly with
respect to the plane of the membrane and releases them
into lipid. We also discuss how the channel would
prevent small molecules from crossing the lipid bilayer
while it is integrating proteins.
Most membrane proteins in eukaryotic cells are integrated into the membrane of the ER before they are
transported in vesicles to the plasma membrane or to
other compartments of the secretory pathway. These
proteins use the same translocation machinery that is
used by secretory proteins [1–6]: namely, a proteinconducting channel formed by the heterotrimeric Sec61
membrane-protein complex. Whereas secretory proteins
cross the membrane completely, only some regions of a
membrane protein are transferred across the membrane;
others remain in the cytosol or stop within the lipid
bilayer. In addition, unlike secretory proteins, membrane
proteins must be oriented: some require that their amino
(N) terminus remains in the cytosol, whereas others need
to have the opposite orientation.
Because membrane proteins can have one or more
transmembrane segments (TMs), the integration machinery must be able to create various topologies, each of which
is somehow dictated by the sequence of the protein. How
membrane proteins integrate into the lipid bilayer and
achieve their different topologies remains mysterious.
What is clear, however, is that most of them integrate into
the ER membrane cotranslationally – in other words,
during their synthesis on membrane-bound ribosomes.
Mechanisms of membrane-protein integration are likely
Corresponding author: Tom A. Rapoport ([email protected]).
Available online 11 September 2004
to be similar in bacteria and archaea because the proteinconducting channel in bacteria is formed by a homolog of
the Sec61 complex, the SecY complex [1,7].
Here, we discuss the mechanism of membrane-protein
integration, with particular reference to the recently
determined crystal structure of the protein-conducting
channel from an archaebacterium [7].
Overview of membrane-protein integration
The integration of a membrane protein begins when a
hydrophobic amino acid sequence emerges from the
translating ribosome and targets the ribosome–nascentchain complex to the ER membrane. The hydrophobic
sequence can be either a signal sequence that will later be
cleaved from the protein or a sequence that will subsequently become a TM. Signal sequences contain relatively short hydrophobic segments and insert into the
Sec61 channel as a loop, with the N terminus remaining in
the cytosol [Figure 1a(i)]. TMs are more hydrophobic than
signal sequences, and whether they insert with the
N terminus staying in the cytosol [Figure 1b(i)] or are
translocated across the membrane [Figure 1c(i)] depends
on the sequence of the protein.
When a TM arrives inside the channel, it must be
recognized and eventually released into the lipid phase
[Figure 1a(iii), 1b(ii,iii), 1c(ii,iii)]. Depending on how a TM
is oriented, during further elongation of the polypeptide
chain, the segment that follows the TM moves either into
the ER lumen [Figure 1b(ii)] or into the cytosol
[Figure 1c(ii)]. When the next TM emerges from the
ribosome (in a multispanning membrane protein), it also
integrates into the lipid bilayer, and the direction of
polypeptide movement switches: if the chain initially
moved into the ER, then it will now move into the cytosol
[Figure 1b(ii,iii)], and vice versa [Figure 1c(ii,iii)]. TMs can
integrate in the order that they are synthesized by the
ribosome, as shown in Figure 1, but the process is often
more complicated.
The molecular mechanisms underlying the processes
depicted in Figure 1 remain unclear, but they place
significant demands on the channel structure. The channel
faces a daunting task: it must open across the membrane
to enable polypeptide segments to cross the lipid bilayer,
and it must let hydrophobic TMs pass through its walls to
enter the lipid. Throughout this process, it must also
prevent small molecules from crossing the membrane. In
www.sciencedirect.com 0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.09.002
Review
TRENDS in Cell Biology
Cleavable
signal sequence
(a)
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(b)
(c)
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Figure 1. Integration of membrane proteins of different topology. (a) Three stages of
integration of a membrane protein with a cleavable signal sequence. (i) The signal
sequence (green) has emerged from the ribosome (blue) and has inserted as a loop
into the Sec61 channel (brown). (ii) At chain elongation, the signal sequence has
been cleaved by signal peptidase (scissors), and a transmembrane segment (TM;
red stripes) is synthesized by the ribosome. (iii) The TM (red) has emerged from the
ribosome, and has left the channel sideways and entered lipid. (b) Three stages of
integration of a membrane protein with a non-cleaved first TM whose N terminus
stays in the cytosol. (i) The first TM has emerged from the ribosome and has
inserted as a loop into the Sec61 channel. (ii) The first TM has left the channel
sideways and entered lipid, and a second TM is synthesized. (iii) The second TM has
emerged from the ribosome, and has left the channel and entered lipid. (c) Three
stages of integration of a membrane protein with a non-cleaved first TM whose
N terminus is translocated. (i) The first TM has emerged from the ribosome and the
N terminus is translocated across the membrane. (ii) The first TM has left the channel
sideways and entered lipid, and a second TM is synthesized. (iii) The second TM has
emerged from the ribosome, and has left the channel and entered lipid.
addition, the channel must be able to orient a TM correctly
and must bind to and dissociate from the ribosome. Until
recently, it was known only that the channel had a
hydrophilic interior [8,9] and a specific binding site for the
signal sequence [10]. How it could perform all of the tasks
demanded of it was difficult to imagine. The crystal
structure of an archaeal channel now suggests some
answers, particularly with regard to how the channel
opens both sideways and from end to end.
Structure of the channel: gating in two directions
When viewed from the cytosol, the channel has a square
shape (Figure 2a). The a-subunit, the largest subunit, has
ten TMs arranged into two domains, TM1–TM5 and TM6–
TM10, which form a ‘clam shell’ hinged by a loop between
TM5 and TM6 at the ‘back’ of the molecule. The two
domains are held together by the g-subunit, which
extends one TM diagonally across the interface between
them and has a second a-helix that lies flat on the
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Vol.14 No.10 October 2004
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cytoplasmic surface of the membrane. The third subunit,
the b-subunit, consists of a single TM.
The b-subunits and g-subunits together occupy three
sides of the a-subunit, leaving unobstructed the side that
is opposite the hinge (the ‘front’ of the complex, where the
mouth of the clamshell would be). The ten helices of
the a-subunit surround a large, water-filled cavity that
has the shape of a funnel that opens on the cytoplasmic
side and extends about halfway across the plane of the
membrane (Figure 2b). The helices on the extracellular
side of the molecule form a similarly shaped cavity, with
the narrow ends of the two cavities meeting in the center
of the structure. On the extracellular side, however, an
additional short helix, the ‘plug’, fills the center of the
cavity (Figure 2b).
The two large, connected cavities in the structure are
likely to form the pore through which proteins pass across
the membrane, with the ‘plug’ acting as a gate. Opening
the pore requires the plug to move into a cavity
present at the back of the molecule (Figures 2a,b). The
open pore has an hourglass shape, with the constriction located in the middle of the membrane. At its
narrowest point, the pore is lined with a single layer of
hydrophobic residues projecting into its center (Figure 2b).
These ‘pore residues’ are thought to fit like a gasket around a
translocating polypeptide. The hourglass shape of the
open channel and the nature of the pore residues
should minimize the channel’s interaction with a
translocating polypeptide chain.
Opening of the pore could be triggered by binding of
both a ribosome and a signal sequence (or a TM). Much
detailed experimental evidence indicates that the hydrophobic core of a signal sequence intercalates between TM2
and TM7 [10], a position that is located at the front of the
channel in the structure. A bound signal sequence would
separate these helices, probably destabilizing the interactions that keep the plug in the center of the molecule.
The segment of the nascent chain that follows the signal
sequence would then occupy the pore, which would remain
open as long as there was a polypeptide within it.
The lateral gate through which hydrophobic segments
of nascent membrane proteins leave the channel and enter
the lipid bilayer is also located at the front of the channel,
at the mouth of the clamshell (Figures 2a and 3). This
conclusion is supported by experimental evidence [10]
and is logical because the structure indicates that it is
the only point where the wall formed by the a-subunit
could open. Opening the lateral gate would require
movement of the hinge at the back of the molecule.
This would separate the helices where the two
domains of the a-subunit touch at the front of the
molecule, creating an opening through which a
segment of nascent chain could pass.
Recognition of TMs in the channel
When inside the channel, a TM of a nascent membrane
protein would encounter both hydrophobic and hydrophilic residues of the lateral gate. This could result in
specific interactions between the TM and the translocation channel. TMs differ widely in sequence, however, and
not all of them could make the same set of interactions. It
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Vol.14 No.10 October 2004
(a)
Plug
γ
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Closed
TM 6–10
α
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chain
Open
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lateral
Signal
sequence
TM 1–5
β
Plug
Open
Perpendicular
(b)
Pore ring
Ribosomebinding site
Cytosol
Plug
Closed
Plug
Pore ring
Open
TRENDS in Cell Biology
Figure 2. Architecture of the translocation channel and proposed mechanism of its gating. (a) Left, top view from the cytosol. Transmembrane segments 1–5 (TM1–TM5) and
6–10 (TM6–TM10) of the a-subunit are shown in blue and red, respectively. The b- and g-subunits are shown in gray. Movement of the plug (yellow) towards the g-subunit is
indicated by the double-headed blue arrow. The loop between TM5 and TM6 could serve as a hinge to open the a-subunit at the front. Right, scheme showing the channel
from the top in the closed state (top), after loop insertion of the signal sequence (middle; the signal sequence is shown in green, the mature part of the nascent chain is shown
in black), and after cleavage of the signal sequence (bottom). The plug has moved towards the back of the a-subunit. (b) Left, cross-sectional view of the closed channel from
the side, with the hydrophobic pore-ring residues shown in green. Right, scheme showing the channel (brown) from the side in its closed (top) and open (bottom) states, the
latter with a nascent chain in the pore and the plug (yellow) at the back of the a-subunit.
therefore seems unlikely that specific interactions with a
TM could function as a general signal to open the lateral
gate. Thus, rather than assuming that TMs have an active
role [6,11,12], it is more attractive to postulate that the
gate ‘breathes’; in other words, it continuously opens and
closes. Such ‘breathing’ would occasionally expose segments of a polypeptide chain located in the aqueous
channel to the hydrophobic interior of the lipid bilayer,
enabling the translocating chain to equilibrate between
the two phases.
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Opening the lateral gate might require little energy
because in the open state the TMs of the channel that form
the gate would no longer be contacted and stabilized by the
plug. In addition, the segments of TM8, TM7, TM2b and
TM3 that contribute to the lateral gate are relatively short
(Figure 3), which might enable parts of the gate to
‘breathe’. Hydrophobic protein segments that are long
enough to span the whole membrane would first intercalate between the TMs that form the lateral gate and
then leave the channel completely by partitioning into the
Review
TRENDS in Cell Biology
TRENDS in Cell Biology
Figure 3. Binding site for signal sequences and lateral gate for transmembrane
segments (TMs) of nascent membrane proteins. A stereo view is shown from the
front, with the TMs of the a-subunit of the channel that contribute to signalsequence binding and to the lateral gate shown in blue (TM2b and TM3) and red
(TM7 and TM8). The hydrophobic core of a signal sequence (green) intercalates in
the cytosolic leaflet of the membrane above the plug (yellow). TMs with longer
hydrophobic segments will exit the lateral gate spanning the whole membrane. The
positions of two arginines and one glutamate, which contribute to the ‘positiveinside rule’ of TM orientation in Saccharomyces cerevisiae, are highlighted in blue
and pink, respectively. The arginines are located at the top of the plug and could
repel the more positively charged flanking region of a TM, whereas the glutamate
residue is located on the cytoplasmic side of the channel and could attract it.
lipid phase [13]. In this view, the channel is needed to
enable a TM to gain access to the hydrophobic interior of
the lipid bilayer. This is supported by studies using a
single-spanning model protein, which have shown that a
TM cannot enter the membrane in the absence of the
Sec61 channel [13], presumably because it cannot cross
the barrier formed by the charged head groups of the
phospholipids. Lateral exit of a TM into lipid seems to be
passive and independent of energy input.
Recent data further support the conclusion that direct
protein–lipid interactions are essential during TM integration mediated by the Sec61 channel. By systematically
incorporating different amino acids into TMs, a ‘biological’
hydrophobicity scale has been established (G. von Heijne,
pers. commun.), which is remarkably similar to the
biophysical one derived from peptide interactions with
model lipid bilayers [14].
When are TMs released into lipid?
Experiments examining the appearance of protein–lipid
crosslinks have shown that a TM comes into contact with
lipids as soon as it is inside the channel [13,15,16]. This is
consistent with the crystal structure, which indicates that
TMs cannot be ‘stored’ within the channel: at most, two
TMs can be present in the channel – one in the pore and
one intercalated into the lateral gate. Thus, during the
synthesis of a multispanning membrane protein, the TMs
could leave the pore one by one or perhaps in pairs. Once a
TM has left the channel, it cannot move far away because
it remains tethered to the ribosome by the following
segment of the nascent chain.
Despite this observation, some TMs that have passed
through the gate apparently have little if any association
with components of the channel. For example, it has been
shown that the TM of one model protein is no longer in
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571
contact with channel proteins and is completely surrounded by lipid long before translation is terminated,
although it is disputed how long the nascent chain must be
before crosslinks to channel components disappear [12,13].
Other crosslinking experiments have shown that TMs
stay close to the channel, sometimes until the termination
of translation [11]. Where are the TMs located in these
cases? One possibility is that they associate with a
‘membrane chaperone’ located at the outside the lateral
gate of the channel.
A candidate for such a chaperone is the translocatingchain-associating membrane (TRAM) protein, a mammalian ER protein that spans the membrane six times [17]
and is located at the front of the Sec61 channel [10,18].
TRAM can be crosslinked to the signal sequences of
secretory proteins and to charged TMs in nascent
membrane proteins [11,13,17], and it is required for the
translocation of secretory proteins with weak signal
sequences [19,20]. TRAM might stabilize less-than-perfect
TMs and facilitate the association of several TMs before
their release as a group into bulk lipid. The bacterial YidC
protein, which has a similar topology to TRAM and is
required for the integration of some membrane proteins,
could have a similar function [21,22].
What determines when a TM is released into bulk lipid?
Hydrophobicity is the simplest possibility: if very hydrophobic, a TM is released early; if less hydrophobic, it stays
close to the channel for an extended time period and might
require association with other TMs for its release into
lipid. Although some data support this model [13], more
recent experiments suggest that factors other than
hydrophobicity – perhaps properties of the sequences
flanking a TM – could also be involved [12]. For multispanning membrane proteins, the integration of a TM
could be facilitated by previously synthesized TMs. For
example, it has been shown for a double-spanning model
protein that a weak second TM, which is unstable in the
lipid phase on its own, is released into bulk lipid upon
interaction with the previously integrated TM [23]; the
latter seems to return to the channel to associate with
the second TM. Other observations in which one TM
requires another for integration might be explained in
a similar way [24,25].
Stability of a TM in a lipid bilayer
Once a TM is in the lipid bilayer, why does it stay there?
The classic view is that membrane proteins are completely
hydrophobic on the surface facing the surrounding lipid.
This view is supported by the crystal structures of many
membrane proteins and by the fact that essentially all
single-spanning membrane proteins have completely
hydrophobic TMs. The concept has been challenged
recently by the proposal that one helix of the
voltage-gated KC channel moves through lipid, even
though it contains four arginines [26]. This raises the
issue of how many charges a TM can contain and still
remain in the membrane.
In vitro experiments in which arginines were placed in
the center of an otherwise hydrophobic TM have shown
that one charged residue does not prevent lateral exit of
the TM from the channel into lipid, but does make the TM
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less likely to remain in the lipid bilayer once it is outside
the channel, as assessed by a lower fraction of integrated
protein and an increase in extractability by alkali [13,23].
The introduction of two arginines leads to a highly
unstable membrane protein. The results were the same
whether the charges were placed in a TM of a single- or
double-spanning membrane protein. These data thus
argue that at most one arginine per TM is tolerable.
How, then, are TMs such as that of the voltage-gated
KC channel inserted into the membrane? These TMs will
probably need to associate with other TMs of the protein to
shield their charges before their release into bulk lipid – a
process that might be facilitated by their transient
association with a ‘membrane chaperone’ such as TRAM.
Orientation of TMs
How can the channel orient a signal sequence or a TM?
Membrane proteins with cleavable signal sequences
always have their newly generated N terminus on the
luminal side [Figure 1a(ii)], whereas single-spanning
membrane proteins with uncleaved signal sequences
that also function as TMs can have two different
orientations (Figure 1b,c).
At least three factors determine the orientation of the
TM. First, the folding characteristics of the N-terminal
domain preceding the TM: only regions without stable
tertiary structure can be translocated [27]. Second, the
distribution of charged amino acids between the segments
flanking the TM: the more positively charged segment
stays in the cytosol (the ‘positive-inside rule’) [28,29].
Third, the length of the hydrophobic sequence: longer
sequences favor localization of the N terminus to the ER
lumen [30,31]. In addition, the orientation of the first TM
of multispanning membrane proteins is often determined
by these factors, and the orientations of the subsequent
TMs alternate correspondingly [32]. As a result, the
topology of the whole protein is determined by its first
TM. There are, however, exceptions where internal TMs
have a preferred orientation regardless of the behavior of
the preceding TMs [33–38].
How can the factors that determine the orientation of
TMs be explained mechanistically? The first is easy to
understand, because a folded N terminus would prevent
its translocation through the narrow pore of the channel.
The second, the positive-inside rule, can be explained, in
part, on the basis of the structure of the channel.
Mutagenesis of two positively charged residues or of a
negatively charged residue in yeast Sec61 has a significant effect on the orientation of a single-spanning reporter
membrane protein [39]. The two positively charged
residues are located on the top of the plug and could
repel the positively charged flanking region of the TM,
whereas the negatively charged residue is located in TM8
and could attract it (Figure 3).
These residues could thus help to orient a TM. They are
unlikely to be the only determinants, however, because the
yeast sec61 mutants are viable and the charged residues
are only well conserved in eukaryotes (although a similar
function might be performed in other species by residues
that occupy spatially adjacent positions). Another mechanism that could contribute to the positive-inside rule is
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Vol.14 No.10 October 2004
the retention of the positively charged flanking region in
the cytosol by its interaction with negatively charged
phospholipid head groups [40]. In bacteria, the membrane
potential might be also involved in orienting a TM [41].
With regard to the third factor, how could a longer
hydrophobic region favor translocation of the N terminus?
Recent experiments in tissue culture cells using a reporter
protein with a TM capable of integration in either
orientation have indicated that it takes time to achieve
the orientation with the N terminus in the cytosol, but the
reverse orientation is attained immediately [42]. A
possible explanation is based on the assumption that all
TMs initially intercalate between the same helices in the
gate that are used by the signal sequences but, because
the hydrophobic sequences in TMs are longer than those
in signal sequences, they can intercalate different regions
of their hydrophobic segments and in either orientation
[Figure 4(i)].
The more hydrophobic the TM, the greater would be its
tendency to partition immediately into the lipid bilayer. If
only the TM has emerged from the ribosome, this would
lead to translocation of the N terminus [(Figure 4(ii)].
Translocation of the N terminus would require a brief
displacement of the plug and would be prevented if the
region preceding the TM is sufficiently positively charged
or folded. Once enough of the segment that follows the TM
has emerged from the ribosome, the carboxyl (C) terminus
could be translocated [Figure 4(iii,iv)]. In this model, TMs
have an opportunity to re-orient by multiple binding and
release events [Figure 4(i)], and they could even invert
their orientation across the membrane [42] [Figure 4(i,ii)].
This would be consistent with the finding that glycosylation of a luminal domain can fix the orientation of a
membrane protein [37], and that downstream TMs of a
multispanning membrane protein can invert a previously
integrated TM [38].
How is the membrane barrier maintained?
Even as a protein is passing through it, the channel must
prevent the passage of small molecules to preserve the
membrane as a barrier. Two models have been proposed to
explain how this is achieved. Using probes to quench
fluorescent molecules incorporated into the translocating
polypeptide chain, Liao et al. [43] have proposed a
sophisticated mechanism by which the membrane
barrier is maintained during the integration of a
single-spanning membrane protein with a cleavable
signal sequence (Figure 5a).
As discussed recently [6], a hallmark of this model is
that a large channel is formed at the interface between
several copies of the heterotrimeric Sec61 complex
(Figure 5a). After being targeted to the membrane by
the signal sequence, the ribosome would make a tight seal
with the cytosolic side of the channel, preventing small
molecules from passing through the junction
[Figure 5a(i)]. Next, the newly synthesized TM, while
still completely inside the ribosome, would form an a-helix
that specifically interacts with some ribosomal proteins
[44] [Figure 5a(ii)]. This interaction would induce a
conformational change in the ribosome that would be
transmitted to the luminal side of the channel, causing
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(ii)
(i)
Cytosol
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N
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site
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N
N
On
go
ing
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elo
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ati
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on
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TRENDS in Cell Biology
Figure 4. Model of the orientation of transmembrane segments (TMs). (i) A hydrophobic TM (red) has emerged from the ribosome (blue) and binds to the signal-binding site
(blue bar) at the front of the channel (brown). The TM can bind reversibly in several conformations and in two different orientations (shown in brackets). (ii) If the hydrophobic
sequence of the TM is long and the N terminus is not retained in the cytosol, it will rapidly flip across the membrane. The plug (yellow) will be displaced only transiently (not
shown). (iii,iv) If the N terminus is retained in the cytosol and the polypeptide chain is elongated further by the translating ribosome, the C terminus can translocate across the
membrane. If a polypeptide chain is present in the pore, the plug will be prevented from returning to its closed-state position.
channel closure through binding of the protein BiP [45]
[Figure 5a(iii)]. Finally, on further extension of the chain
by two amino acids, another conformational change would
open the ribosome–channel junction. Because the ribosome stays bound to the channel [16], it would detach only
at one side, creating a path for the polypeptide chain into
the cytosol [Figure 5a(iv)].
This model raises several questions. How could the
ribosomal tunnel recognize the wide range of TM
sequences and distinguish them from signal sequences,
given that it does not contain extensive hydrophobic
surfaces [46]? How could the tunnel bind TMs and yet not
prevent them from moving on? How could the next TM
induce gating changes in the reverse order, even though it
passes the ribosomal proteins in the same direction? How
can BiP provide a luminal seal but still allow movement of
a polypeptide into the ER? Clearly, more experiments,
including studies using techniques other than fluorescence quenching, are required to answer these and
other questions. It should be also noted that this model
does not explain how the membrane barrier is maintained
during posttranslational translocation, in the absence of a
ribosome or, in bacteria, in the absence of BiP.
An alternative explanation of how the passage of small
molecules through the channel is hindered during membrane-protein integration is suggested by structural
studies. Electron cryo-microscopy indicates that there is
always a gap between the ribosome and the channel [47–50].
This gap (12–15 Å) is consistent with the size of the
cytosolic loops in the C-terminal half of Sec61, which
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probably provide the principal ribosome-binding sites [51].
Its width is sufficient for the exit of a polypeptide segment
into the cytosol, but small enough to explain the
inaccessibility of a translocating nascent chain to proteases [52]. It might also explain why IK ions, used as a
fluorescence quencher, cannot reach probes in the nascent
chain (hydrated IK ions have a diameter of about 8 Å and
also might be repelled by negative charges of rRNA
pointing into the gap).
The open ribosome–channel junction would not compromise the membrane barrier, because the crystal
structure of the channel suggests that the membrane
barrier is maintained inside the channel (Figure 5b).
According to the crystal structure, only one copy of the
Sec61 complex forms an active pore, even though
additional complexes are associated with the ribosome
during translocation [47–50] (Figure 5b). When a segment
of a nascent membrane protein is located in the pore, the
plug will be at the back of the channel and the ring of pore
residues will form a seal around the polypeptide chain
[Figure 5b(i,ii)]. Although the seal is not likely to be
perfect, it will significantly hinder the membrane permeation of small molecules during translocation.
Once the TM has moved laterally out of the channel, the
plug could return to its closed-state position and block the
passage of ions and other small molecules [Figure 5b(iii)].
This simple mechanism would ensure that, at all times,
either the nascent chain or the plug blocks the pore and
maintains the membrane barrier. This model would be
applicable to all modes of translocation in all organisms.
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(a)
(b)
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orientation and assemble with other TMs, and how the
membrane barrier is maintained. New methods are
required to follow the integration of individual TMs
during the synthesis of multispanning membrane proteins, to probe the localization of luminal and cytosolic
segments, to test the folding of membrane proteins, and to
identify membrane chaperones. The crystal structure of
the channel has led to simple models of membrane-protein
integration and provides an excellent basis on which to
test them.
(ii)
Acknowledgements
N
We thank Bil Clemons for help with the figures, and Andrew Osborne and
Bert van den Berg for critically reading the manuscript.
N
References
TM
(iii)
TM
BiP
N
N
(iv)
N
N
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Figure 5. Two models of the maintenance of the membrane barrier during the
synthesis of membrane proteins. (a) The flow of small molecules through the
membrane is prevented on the cytosolic side by the channel-bound ribosome and,
on the luminal side, by BiP. (i) A signal sequence (green) has emerged from the
ribosome (blue) and has inserted as a loop into the channel (brown). The ribosome
makes a tight seal with the Sec61 channel, preventing small molecules in the
cytosol and endoplasmic reticulum lumen (green and red dots) from passing the
membrane. In this model, a large membrane channel is formed at the interface
between several copies of the Sec61 complex (two copies are shown in crosssection). (ii) The signal sequence has been cleaved by signal peptidase (scissors).
(iii) A TM (red stripes) is synthesized by the ribosome, causing a conformational
change on the luminal side of the channel, which triggers the binding of BiP (blue).
(iv) The TM (red) has left the channel sideways and entered lipid, the ribosome is
partially detached from the channel, and the seal is now provided on the luminal
side by BiP. (b) The membrane barrier is maintained inside the channel, either by
the plug domain or by the translocating chain located in the pore. (i) The signal
sequence is bound to the channel and the pore has opened by movement of the
plug (yellow). The pore-inserted region of the nascent chain that follows the signal
sequence prevents the plug from returning to its closed-state position and
obstructs small molecules from passing the membrane. In this model, a single
copy of the Sec61 complex forms an active pore, although additional copies are
associated with the ribosome. (ii) The signal sequence has been cleaved. (iii) The
TM has left the channel sideways and entered lipid, the polypeptide segment that
follows emerges into the cytosol through a gap between the ribosome and channel,
and the plug has returned to its closed-state position, preventing small molecules
from passing the membrane. (iv) A second TM has been synthesized by the
ribosome, and the following polypeptide segment is now located in the open pore.
Concluding remarks
It is apparent that our understanding of how membrane
proteins integrate is still rudimentary and that major
points of disagreement in the field must be resolved.
Questions that need answers relate to the stage when TMs
are released into bulk lipid, how TMs achieve their
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