Báo cáo khoa học: Protein transport across the endoplasmic reticulum membrane pdf

THE SIR HANS KREBS LECTURE
Protein transport across the endoplasmic reticulum
membrane
Delivered on 8 July 2007 at the 32nd FEBS Congress in Vienna,
Austria
Tom A. Rapoport
Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, MA, USA
Introduction
Protein translocation across the eukaryotic endopl-
asmic reticulum (ER) membrane is a decisive step in
the biosynthesis of many proteins [1]. These include
soluble proteins, such as those ultimately secreted from
the cell or localized to the ER lumen, and membrane
proteins, such as those in the plasma membrane or in
other organelles of the secretory pathway. Soluble
proteins cross the membrane completely and usually
have cleavable N-terminal signal sequences, whose
major feature is a segment of approximately seven to
12 hydrophobic amino acids. Integral membrane
proteins have one or more transmembrane (TM)
segments, each containing approximately 20 hydropho-
bic amino acids, with intervening hydrophilic regions
on either side of the membrane. Both types of proteins
use the same machinery for transport across the
membrane: a protein-conducting channel. This channel
allows polypeptides to cross the membrane and per-
mits hydrophobic TM segments of membrane proteins
to exit laterally into the lipid phase. In bacteria, the
translocation of secretory and membrane proteins
occurs through a homologous channel in the plasma
membrane, employing signal and TM sequences that

are similar to those in eukaryotes.
The translocation channel is formed from an evolu-
tionarily conserved heterotrimeric membrane protein
Keywords
endoplasmic reticulum; lipid; membrane
integration; protein-conducting channel;
protein translocation; ribosome; Sec
complex; Sec61; SecA; SecY
Correspondence
T. A. Rapoport, Howard Hughes Medical
Institute and Department of Cell Biology,
Harvard Medical School, 240 Longwood
Avenue, Boston, MA 02115, USA
Fax: +1 617 432 1190
Tel: +1 617 432 0676
E-mail:
(Received 21 May 2008, accepted 18 June
2008)
doi:10.1111/j.1742-4658.2008.06588.x
A decisive step in the biosynthesis of many eukaryotic proteins is their par-
tial or complete translocation across the endoplasmic reticulum membrane.
A similar process occurs in prokaryotes, except that proteins are trans-
ported across or are integrated into the plasma membrane. In both cases,
translocation occurs through a protein-conducting channel that is formed
from a conserved, heterotrimeric membrane protein complex, the Sec61 or
SecY complex. Structural and biochemical data suggest mechanisms that
enable the channel to function with different partners, to open across the
membrane and to release laterally hydrophobic segments of membrane
proteins into lipid.
Abbreviations

EM, electron microscopy; ER, endoplasmic reticulum; SRP, signal recognition particle; TM, transmembrane.
FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS 4471
complex, called the Sec61 complex in eukaryotes and
the SecY complex in bacteria and archaea [2]. The
a-subunit forms the channel pore, as originally demon-
strated by systematic crosslinking experiments [3]. In
addition, reconstitution experiments show that the
Sec61 ⁄ SecY complex is the only essential membrane
component for protein translocation in mammals and
bacteria [4–6]. The channel has an aqueous interior,
as demonstrated by electrophysiology experiments
and measurements of the fluorescence life-time of
probes incorporated into a translocating polypeptide
chain [7–9].
Different modes of translocation
The channel must associate with partners that provide
the driving force for translocation. Depending on the
channel partner, there are different known transloca-
tion modes [1]. In co-translational translocation, the
major partner is the ribosome. This translocation
mode is found in all cells of all species, and it is used
for the translocation of both secretory and membrane
proteins. Co-translational translocation begins with a
targeting phase, during which a ribosome-nascent
chain complex is directed to the membrane by the
signal recognition particle (SRP). The ribo-
some ⁄ SRP ⁄ nascent chain complex is bound to the
membrane, first by an interaction between SRP and its
membrane receptor (SR), and then by an interaction
between the ribosome and the translocation channel

(Fig. 1). The elongating polypeptide chain sub-
sequently moves directly from a tunnel inside the ribo-
some into the associated membrane channel. GTP
hydrolysis during translation provides the energy for
translocation.
In most, if not all cells, some proteins are translocated
after their completion. This post-translational translo-
cation occurs by different mechanisms in eukaryotes
and bacteria. In yeast (and probably in all eukaryotes),
translocation occurs by a ratcheting mechanism and
involves, as channel partners, the tetrameric Sec62⁄ 63
membrane protein complex and the ER luminal
protein BiP, a member of the Hsp70 family of ATPases
(Fig. 2) [10]. Following a transient interaction of BiP-
ATP with the J-domain of Sec63p, ATP is hydrolyzed
and the peptide-binding pocket of BiP closes around
the translocation substrate. BiP serves as a Brownian
ratchet, preventing the bound polypeptide from sliding
SRP receptor
ribosome
SRP
Sec61/SecY
complex
signal
sequence
cytosol
Fig. 1. Model of co-translational trans-
location. The polypeptide chain moves from
the tunnnel inside the ribosome into the
membrane channel. The energy for

translocation is provided by GTP hydrolysis
during translation. Figure adapted from [1].
Sec61
complex
ADP ADP
ADP
ADP
ADP
ADP
ATP
ATP
ATP
cytosol
ATP
Sec62/63
complex
signal
sequence
BiP
J-domain
Fig. 2. Model of post-translational transloca-
tion in eukaryotes. A Brownian ratcheting
mechanism is responsible for moving a poly-
peptide chain through the membrane. Trans-
location might be mediated by oligomers of
the Sec61p complex, as in the other modes
of translocation. Figure adapted from [1].
Protein transport across the ER membrane T. A. Rapoport
4472 FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS
back into the cytosol, but allowing polypeptide move-

ment in the forward direction. When moved suffi-
ciently, the next BiP molecule binds, and this process
is repeated until the entire polypeptide chain is translo-
cated. Finally, nucleotide exchange of ADP for ATP
opens the peptide binding pocket and releases BiP
from the polypeptide.
In eubacterial post-translational translocation, poly-
peptides are ‘pushed’ through the channel by its part-
ner, the cytosolic ATPase SecA (Fig. 3) [11]. SecA has
two nucleotide binding domains (NBD1 and NBD2),
which bind the nucleotide between them and move
relative to one another during the ATP hydrolysis
cycle. Exactly how these movements are used to ‘push’
a polypeptide chain through the channel remains
unknown. The size of SecA makes it unlikely that it
inserts deeply into the SecY channel, as proposed pre-
viously [11]. Bacterial translocation in vivo requires an
electrochemical gradient across the membrane, but the
mechanism by which the gradient is utilized is unclear.
Archaea probably have both co- and post-transla-
tional translocation. Although co-translational trans-
location is likely to be similar to that in eukaryotes
and eubacteria, it is not known how post-translational
translocation occurs because archaea lack SecA, the
Sec62 ⁄ 63 complex and BiP.
Structure and function of the
translocation channel
The crystal structure of an archaeal SecY complex pro-
vides much insight into channel function [2]. The struc-
ture is likely representative of all species, as indicated

by sequence conservation and by the similarity to a
lower resolution structure of the Escherichia coli SecY
complex determined by electron microscopy (EM)
from 2D crystals [12]. The a-subunit consists of two
halves, TMs 1–5 and TMs 6–10, which form a lateral
gate at the front and are clamped together at the back
by the c-subunit (Fig. 4A). The 10 helices of the a-sub-
unit form an hourglass-shaped pore that consists of
cytoplasmic and external funnels whose tips meet
approximately half way across the membrane
(Fig. 4B). The cytoplasmic funnel is empty, whereas
the external funnel is filled by a short helix, the ‘plug’.
The constriction of the hourglass is formed by a ‘pore

signal
sequence
NBF1
ADP
ATP
SecY
complex
SecA
cytosol
Fig. 3. Model of post-translational transloca-
tion in bacteria. The ATPase SecA uses the
energy of ATP hydrolysis to push a poly-
peptide through the channel. Figure adapted
from [1].
lateral gate
β

γ
pore
ring
pore
ring
A
B
cytosol
hinge
plug
plug
back
front
Fig. 4. Structure of the translocation channel. (A) View from the
cytosol of the X-ray structure of the SecY complex from Methano-
coccus jannaschii. The two halves of SecY are colored blue (TM
1–5) and red (TM 6–10). The plug is shown in yellow and pore ring
residues are shown in green. The purple arrow indicates how the
lateral gate opens. The black arrow indicates how the plug moves
to open the channel across the membrane. (B) Cross-section of the
channel from the side. Figure adapted from [1].
T. A. Rapoport Protein transport across the ER membrane
FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS 4473
ring’ of hydrophobic amino acid residues that project
their side chains radially inward. The crystal structure
represents a closed state of the channel, but biochemi-
cal data indicate how it can open to translocate
proteins (see below).
Opening the channel across the
membrane

The crystal structure indicates that a single copy of the
Sec61 ⁄ SecY complex forms the pore [2]. The transloca-
tion of a secretory protein begins with insertion of a
loop into the channel, such that the signal sequence
is intercalated into the walls of the channel and the
segment distal to it is inserted into the pore proper
(Figs 1–3). In a first step, the binding of a channel
partner (i.e. the ribosome, the Sec62⁄ 63p complex, or
SecA) likely weakens interactions that keep the plug in
the center of the Sec61 ⁄ SecY molecule, as indicated by
an increased ion conductance when nontranslating
ribosomes are bound to the channel [7]. Next, the
hydrophobic segment of a signal sequence intercalates
into the lateral gate, between TM2b and TM7, as indi-
cated by photo-crosslinking experiments [13]. This fur-
ther destabilizes plug interactions, causing the plug to
be displaced from the center of Sec61 ⁄ SecY, as shown
by disulfide bridge crosslinking [14,15]. During sub-
sequent translocation, the signal sequence remains
stationary, whereas the rest of the polypeptide moves
through the pore from the cytoplasmic funnel through
the pore ring into the extracellular funnel (Figs 1–3),
as indicated by systematic disulfide crosslinking experi-
ments [16]. The aqueous interior of the channel and its
shape help to minimize the energy required for the
translocation of a polypeptide through the membrane.
The plug can only return to the center of Sec61 ⁄ SecY
when the polypeptide chain has left the pore.
The diameter of the pore ring, as observed in the
crystal structure, has to increase during translocation,

probably by movements of the helices to which the
pore ring residues are attached. The pore ring is indeed
flexible, as shown by molecular dynamics simulations
and electrophysiology experiments [17,18]. The maxi-
mum size of the pore could be 15 · 20 A
˚
, which is
much smaller than the pore size estimated from fluo-
rescence quenching experiments (40–60 A
˚
) [19]. These
data could be reconciled with the crystal structure if
two or more Sec61 ⁄ SecY complexes associated at their
front surfaces, opened their lateral gates, and fused
their pores to form a larger channel. However, disul-
fide bridge crosslinking experiments argue against
fusion of different pores because they show that,
during SecA-mediated translocation, both the signal
sequence and the mature region of a polypeptide chain
are located in the same SecY molecule [20]. In addi-
tion, a detergent-solubilized translocation intermediate
also contains just one copy of SecY associated with
one SecA and one translocation substrate molecule
[21]. Two SecY molecules in a nearly front-to-front
orientation were proposed to be associated with a
translating E. coli ribosome [22]. However, this conclu-
sion was based on a low-resolution ( 15 A
˚
)EM
structure, and the docking of the crystal structure

required its drastic modification. Furthermore, the
position and orientation of both SecY molecules are
different from that of the single SecY molecule
observed in recent EM structures of nontranslating
ribosome-SecY complexes [23] (Fig. 5). It should be
noted that, in the fluorescence quenching experiments,
the fluorescent probes were located deep inside the
ribosome, and therefore the same large diameter
(40–60 A
˚
) must be assumed for the ribosome tunnel, a
size that does not agree with that seen in ribosome
structures (< 20 A
˚
) determined by crystallography
or cryo-EM [24]. Taken together, it is likely that the
translocation pore is formed by just one copy of the
Sec61 ⁄ SecY complex.
Oligomeric translocation channels
Although the pore is formed by only one Sec61 ⁄ SecY
molecule, translocation of a polypeptide chain appears
to be mediated by oligomers. This conclusion is based
on the observation that a SecY molecule defective in
SecA-mediated translocation can be rescued by linking
it covalently with a wild-type SecY copy [20]. Disulfide
bridge crosslinking showed that SecA interacts through
its NBD1 with a nontranslocating SecY copy and
moves the polypeptide chain through a neighboring
SecY copy. The Sec61⁄ SecY complex probably forms
oligomers during co-translational translocation as well.

When a ribosome ⁄ nascent chain ⁄ SRP complex binds
to the SRP receptor, a domain of SRP undergoes a
conformational change, exposing a site on the ribo-
some to which a single Sec61 ⁄ SecY molecule could
bind [25]. This is likely to be the molecule seen in
recent EM structures of complexes of nontranslating
ribosomes with either the SecY or the Sec61 complex
[23,23a]. The bound SecY ⁄ Sec61 molecule is close to
the point where a polypeptide exits the ribosome
and could thus become the translocating copy (Fig. 5).
At a later stage of translocation, SRP completely
detaches from the ribosome, and an additional copy
of the Sec61 ⁄ SecY complex may associate (Fig. 1),
as suggested by crosslinking and freeze-fracture EM
experiments [26,27]. These copies could stabilize the
Protein transport across the ER membrane T. A. Rapoport
4474 FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS
ribosome–channel junction and possibly recruit other
components, such as signal peptidase and oligosaccha-
ryl transferase, or the translocon-associated protein
complex. Upon termination of translocation, disso-
ciation of the Sec61 ⁄ SecY oligomers could facilitate
the release of the ribosome from the membrane.
Dissociable oligomers may also allow the Sec61 ⁄ SecY
complex to change channel partners and modes of
translocation.
The EM structures of detergent-solubilized ribo-
some–channel complexes suggested the presence of
three or four Sec61 molecules [28,29]. However, it now
appears that only one Sec61 molecule is present and

that the additional density can be attributed to lipid
and ⁄ or detergent.
Membrane protein integration
During the synthesis of a membrane protein, hydro-
phobic TM segments move from the aqueous interior
of the channel through the lateral gate into the lipid
phase. The lateral gate may continuously open and
close, exposing polypeptide segments located in the
aqueous channel to the surrounding hydrophobic lipid
phase. Alternatively, there may be a ‘window’ in the
lateral gate that would allow the hydrocarbon chains
of lipids to make contact with a translocating polypep-
tide at the same time as preventing charged head
groups from entering the channel. Polypeptide seg-
ments inside the channel would partition between the
aqueous and hydrophobic environments. This model is
supported by photo-crosslinking experiments [30] and
by the close correlation between a hydrophobicity scale
and the tendency of a peptide to span the membrane
[31]. Hydrophilic segments between the TMs would
alternately move from the ribosome through the aque-
ous channel to the external side of the membrane, or
emerge into the cytosol between the ribosome and
channel through a ‘gap’ that can be visualized in EM
structures [28,29].
The first TM segment of a membrane protein can
have its N-terminus on either side of the membrane,
depending on the amino acid sequence of the protein,
which often determines the orientation of subsequent
TMs. If the first TM is long and the preceding

sequence not retained in the cytosol by positive
charges or by its folding, the N-terminus can flip
across the channel and subsequently exit laterally into
the lipid phase. When the N-terminus is retained in the
cytosol and the polypeptide chain is further elongated,
SecY
A
B
tunnel
exit
Fig. 5. Structure of the E. coli ribosome-associated SecY channel. (A) Bottom view showing the single copy of the SecY complex that is
bound to a nontranslating ribosome. The docked X-ray structure is indicated by the ribbons. The electron density of the channel is shown in
transparent pink. (B) Comparison of the single-SecY model (left) with a model [22] in which two SecY copies are bound in a near front-to-
front orientation to a translating ribosome (right). The lateral gate of the SecY channel is indicated by an arrow, and the tunnel exit by a star.
The smaller picture above shows the orientation of the ribosome.
T. A. Rapoport Protein transport across the ER membrane
FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS 4475
the C-terminus can translocate across the channel,
inserting the polypeptide as a loop, as in the case of a
secretory protein.
Maintaining the permeability barrier
The channel must prevent the free movement of small
molecules, such as ions or metabolites. The crystal
structure suggests a simple model for the maintenance
of the membrane barrier. The resting channel would
be closed, which is consistent with electrophysiology
experiments showing that, in the absence of other com-
ponents, the SecY channel is impermeable to ions and
water [18]. In the active channel, the pore ring would
fit around the translocating polypeptide chain like a

gasket to restrict the passage of small molecules. The
seal would not be expected to be perfect, but leakage
could be compensated for by powerful ion pumps.
During the synthesis of a multi-spanning membrane
protein, the seal would be provided in an alternating
manner by either the nascent chain in the pore or,
once the chain has left the pore, by the plug returning
to the center of Sec61⁄ SecY. Although this model
needs further experimental verification, it would
explain how the membrane barrier can be maintained
in both co- and post-translational translocation.
Surprisingly, plug deletion mutants are viable in
Saccharomyces cerevisiae and E. coli and have only
moderate translocation defects [32–34]. However, the
crystal structures of these mutants show that new plugs
are formed from neighboring polypeptide segments
[34]. The new plugs still seal the closed channel, but
they have lost many interactions that normally keep
the plug in the center of SecY. This results in continu-
ous channel opening and closing, and permits polypep-
tides with defective or even missing signal sequences to
be translocated. The plug sequences are only poorly
conserved among Sec61 ⁄ SecY channels, supporting the
idea that promiscuous segments can seal the channel
and lock it in its closed state.
Perspective
We are beginning to understand protein translocation
across the eukaryotic ER and bacterial plasma mem-
branes at the molecular level. In particular, progress
during the recent years has led to important insights

into the function of the Sec61 ⁄ SecY channel. Neverthe-
less, there are major questions in the field that remain
controversial and unresolved, and further progress will
require a combination of approaches. Electrophysi-
ology experiments are needed to complement the fluo-
rescence quenching method, particularly because the
results obtained from the latter are difficult to recon-
cile with structural data. Important questions with
respect to co-translational translocation include how
the SRP receptor and channel collaborate, how many
Sec61 ⁄ SecY complexes participate in translocation,
and how the ribosome ultimately dissociates from the
channel. Both the precise role of the Sec62 ⁄ 63 compo-
nents in post-translational translocation and the mech-
anism by which SecA moves polypeptides need to be
clarified. Membrane protein integration is still particu-
larly poorly understood, and new methods are required
to follow the membrane integration of TMs. Several
other translocation components have been identified,
such as the TRAM protein and the translocon-associ-
ated protein complex in mammalian cells, or the YidC
and SecDF proteins in prokaryotes. These components
may be required as chaperones for the folding of TM
segments, or to increase the efficiency of translocation
of some substrates, but their precise functions remain
to be clarified. Much of the progress in the field will
hinge on structural data, with the ‘holy grail’ being
a picture of an active translocon, where a channel
associated with both a partner and a translocating
polypeptide chain is visualized at the atomic level. The

results obtained will likely serve as a paradigm for
other protein translocation systems, such as those in
mitochondria, chloroplasts and peroxisomes.
Acknowledgements
Work in the author’s laboratory was supported by
grants from the National Institute of Health. The
author is a Howard Hughes Medical Institute Investi-
gator. Briana Burton and Sol Schulman are thanked
for critically reading the manuscript.
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