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: tom_rapoport@hms.harvard.edu (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. References 1 Rapoport TA (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450 , 663–669. 2 van den Berg B, Clemons WM Jr, Collinson I, Modis Y, Hartmann E, Harrison SC & Rapoport TA (2004) X-ray structure of a protein-conducting channel. Nat- ure 427, 36–44. 3 Mothes W, Prehn S & Rapoport TA (1994) Systematic probing of the environment of a translocating secre- tory protein during translocation through the ER membrane. EMBO J 13, 3937–3982. 4 Akimaru J, Matsuyama SI, Tokuda H & Mizushima S (1991) Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Esc- herichia coli. Proc Natl Acad Sci USA 88, 6545–6549. Protein transport across the ER membrane T. A. Rapoport 4476 FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS 5 Brundage L, Hendrick JP, Schiebel E, Driessen AJM & Wickner W (1990) The purified E. coli integral membrane protein SecY ⁄ E Is sufficient for reconstitu- tion of SecA-dependent precursor proteintranslocation. Cell 62, 649–657. 6Go ¨ rlich D & Rapoport TA (1993) Protein transloca- tion into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630. 7 Simon SM & Blobel G (1991) A protein-conducting channel in the endoplasmic reticulum. Cell 65, 371– 380. 8 Crowley KS, Liao SR, Worrell VE, Reinhart GD & Johnson AE (1994) Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461–471. 9 Crowley KS, Reinhart GD & Johnson AE (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115. 10 Matlack KE, Misselwitz B, Plath K & Rapoport TA (1999) BiP acts as a molecular ratchet during post- translational transport of prepro-a factor across the ER membrane. Cell 97, 553–564. 11 Economou A & Wickner W (1994) SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell 78, 835–843. 12 Breyton C, Haase W, Rapoport TA, Kuhlbrandt W & Collinson I (2002) Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662–665. 13 Plath K, Mothes W, Wilkinson BM, Stirling CJ & Rapoport TA (1998) Signal sequence recognition in posttranslational protein transport across the yeast ER membrane. Cell 94, 795–807. 14 Harris CR & Silhavy TJ (1999) Mapping an interface of SecY (PrlA) and SecE (PrlG) by using synthetic phenotypes and in vivo cross-linking. J Bacteriol 181, 3438–3444. 15 Tam PC, Maillard AP, Chan KK & Duong F (2005) Investigating the SecY plug movement at the SecYEG translocation channel. EMBO J 24, 3380–3388. 16 Cannon KS, Or E, Clemons WM Jr, Shibata Y & Rapoport TA (2005) Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY. J Cell Biol 169, 219–225. 17 Gumbart J & Schulten K (2006) Molecular dynamics studies of the archaeal translocon. Biophys J 90, 2356– 2367. 18 Saparov SM, Erlandson K, Cannon K, Schaletzky J, Schulman S, Rapoport TA & Pohl P (2007) Deter- mining the conductance of the SecY protein trans- location channel for small molecules. Mol Cell 26, 501–509. 19 Hamman BD, Chen JC, Johnson EE & Johnson AE (1997) The aqueous pore through the translocon has a diameter of 40-60A ˚ during cotranslational protein translocation at the ER membrane. Cell 89, 535–544. 20 Osborne AR & Rapoport TA (2007) Protein trans- location is mediated by oligomers of the SecY complex with one SecY copy forming the channel. Cell 129, 97–110. 21 Duong F (2003) Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase. EMBO J 22 , 4375–4384. 22 Mitra K, Schaffitzel C, Shaikh T, Tama F, Jenni S, Brooks CL 3rd, Ban N & Frank J (2005) Structure of the E. coli protein-conducting channel bound to a translating ribosome. Nature 438, 318–324. 23 Menetret JF, Schaletzky J, Clemons WM Jr, Osborne AR, Skanland SS, Denison C, Gygi SP, Kirkpatrick DS, Park E, Ludtke SJ et al. (2007) Ribosome binding of a single copy of the SecY complex: implications for protein translocation. Mol Cell 28, 1083–1092. 23a Menetret JF, Hedge RS, Aguiar M, Gygi SP, Park E, Rapaport TA & Akey CW (2008) Single copies of Sec61 and TRAP associate with a nontranslating mammalian ribosome. Structure 16, 1126–1137. 24 Voss NR, Gerstein M, Steitz TA & Moore PB (2006) The geometry of the ribosomal polypeptide exit tunnel. J Mol Biol 360, 893–906. 25 Halic M, Gartmann M, Schlenker O, Mielke T, Pool MR, Sinning I & Beckmann R (2006) Signal recogni- tion particle receptor exposes the ribosomal translocon binding site. Science 312, 745–747. 26 Schaletzky J & Rapoport TA (2006) Ribosome bind- ing to and dissociation from translocation sites of the endoplasmic reticulum membrane. Mol Biol Cell 17 , 3860–3869. 27 Hanein D, Matlack KES, Jungnickel B, Plath K, Kalies K-U, Miller KR, Rapoport TA & Akey CW (1996) Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87, 721–732. 28 Beckmann R, Spahn CM, Eswar N, Helmers J, Pen- czek PA, Sali A, Frank J & Blobel G (2001) Architec- ture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361–372. 29 Menetret JF, Hegde RS, Heinrich SU, Chandramouli P, Ludtke SJ, Rapoport TA & Akey CW (2005) Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 348, 445–457. 30 Heinrich SU, Mothes W, Brunner J & Rapoport TA (2000) The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102, 233–244. T. A. Rapoport Protein transport across the ER membrane FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS 4477 31 Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H, Nilsson I, White SH & von Heijne G (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381. 32 Junne T, Schwede T, Goder V & Spiess M (2006) The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability. Mol Biol Cell 17, 4063–4068. 33 Maillard AP, Lalani S, Silva F, Belin D & Duong F (2007) Deregulation of the SecYEG translocation channel upon removal of the plug domain. J Biol Chem 282, 1281–1287. 34 Li W, Schulman S, Boyd D, Erlandson K, Beckwith J & Rapoport TA (2007) The plug domain of the SecY protein stabilizes the closed state of the translocation channel and maintains a membrane seal. Mol Cell 26, 511–521. Protein transport across the ER membrane T. A. Rapoport 4478 FEBS Journal 275 (2008) 4471–4478 ª 2008 The Author Journal compilation ª 2008 FEBS . 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 USA Introduction Protein translocation across the eukaryotic endopl- asmic reticulum (ER) membrane is a decisive step in the biosynthesis of many proteins [1]. These