retro-translocation of proteins from the endoplasmic reticulum into the cytosol

246 | APRIL 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

R E V I E W S

Many proteins are synthesized in the cytosol andtranslocated across, or integrated into, the endoplas-mic reticulum (ER) membrane; for example, secre-tory proteins, plasma membrane proteins, proteins ofthe ER, of the Golgi apparatus, and of otherorganelles that are derived from the secretory path-way. The mechanism of their translocation from thecytosol into and across the ER membrane has beenextensively studied and important aspects have beenclarified (for review, see REF. 1). In recent years, how-ever, it has become clear that there is a translocationpathway in the reverse direction — called retro-translocation — which results in the export of pro-teins from the lumen or membrane of the ER into thecytosol. Unlike ‘forward translocation’, this pathway isonly poorly understood.

Substrates of the retro-translocation pathwayMisfolded or misassembled ER proteins. When newlysynthesized proteins emerge from the cytosol into theER lumen, they undergo ‘quality control’. An elaboratesystem of CHAPERONES assists proteins in their foldingand prevents misfolded proteins from reaching theirfinal destination, such as the cell surface. Chaperonesalso catalyse the oligomeric assembly of protein com-plexes before they exit the ER. This is particularly rele-vant for membrane proteins, most of which seem tobe oligomeric.

When proteins misfold in the ER, they elicit the‘unfolded protein response’(UPR) (for review,see REF.2).Asa result of signalling from the ER lumen to the cell nucleus,the transcriptional expression of chaperones is stimulated,which results in higher chaperone concentrations in theER lumen, and helps to rectify the folding problem.However, proteins cannot always be brought back to thenative state and these polypeptides are degraded. In fact,the UPR also results in the induction of components thatare required for degradation,and,vice versa3–5.

Although some misfolded proteins are transportedto and degraded in the LYSOSOME 6,7, most seem to behandled at the ER. It was initially thought that proteol-ysis occurs inside the membrane or lumen of the ER(for review of the early work, see REF. 8). However, theeffect of mutants in, and inhibitors of, theubiquitin–proteasome system (BOX 1) later showed thatdegradation occurs in the cytosol (for review, see REF. 9).So, a new model emerged in which misfolded ER sub-strates — both lumenal and transmembrane — aredisposed of by the same machinery that is responsiblefor the removal of misfolded cytosolic proteins.Obviously, the ER proteins must first be retro-translo-cated or ‘dislocated’ into the cytosol before they can beattacked by the ubiquitin–proteasome system. Thepathway is often referred to as ‘ER-associated degrada-tion’ (ERAD)10. Substrates of the pathway also includesome wild-type proteins, such as the cystic fibrosis

RETRO-TRANSLOCATION OFPROTEINS FROM THEENDOPLASMIC RETICULUM INTO THE CYTOSOLBilly Tsai*, Yihong Ye* and Tom A. Rapoport

Proteins that are misfolded in the endoplasmic reticulum are transported back into the cytosol fordestruction by the proteasome. This retro-translocation pathway has been co-opted by certainviruses, and by plant and bacterial toxins. The mechanism of retro-translocation is stillmysterious, but several aspects of this process are now being unravelled.

CHAPERONES

A class of proteins that bindunfolded or partially foldedpolypeptides, prevent theiraggregation and promote theirfolding.

LYSOSOME

An organelle (called the vacuolein yeast) that contains manyhydrolytic enzymes. Lysosomesdegrade proteins that areimported into the cell byendocytosis, which are divertedto them from the secretorypathway, or taken up from thecytosol by autophagy.

Howard Hughes MedicalInstitute and Department of Cell Biology, HarvardMedical School, Boston,MA 02115 USA. *Theseauthors contributed equally.Correspondence to T.A.R.e-mail: [email protected]: 10.1038/nrm780

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | APRIL 2002 | 247

R E V I E W S

INCLUSION BODIES

Protein precipitates that areformed in the cytosol, oftenafter protein overexpression.

the misfolded asialoglycoprotein receptor was found toaccumulate at the centre of cells after treatment withproteasome inhibitors18.

Co-opting the cellular system of retro-translocation. TheER quality-control system might be co-opted by someviruses. The expression of certain viral proteins leads tothe selective degradation of normal cellular proteins thatare required for the immune defense of the host. Forexample, the expression of either US2 or US11 — twoproteins of the human cytomegalovirus (HCMV) —targets the newly synthesized major histocompatibility(MHC) class I heavy chain for degradation19,20. This sin-gle-spanning protein is recognized by the virally encodedproteins, exported from the ER into the cytosol anddegraded by the ubiquitin–proteasome system. The Vpuprotein of the human immunodeficiency virus (HIV) alsotriggers ER retention and degradation of the CD4receptor21, and the MK3 protein of the murine γ-her-pesvirus-68 leads to the selective degradation of MHCclass I heavy chains by the ubiquitin–proteasome system22.

The same pathway of retro-translocation might alsobe used by certain toxins, including cholera toxin, entero-toxin and ricin23,24. Cholera toxin, for example, is takenup by endocytosis and transported backwards throughthe secretory pathway until it arrives in the ER lumen. Afragment of the toxin is then released from the rest of themolecule and is retro-translocated across the ER mem-brane into the cytosol25,26. In contrast to the other sub-strates discussed above, the toxin fragment is notdegraded, at least not completely. It becomes an activeenzyme that modifies a heterotrimeric G protein, which,in turn, leads to the opening of a chloride channel withconsequent massive chloride and water secretion.

A general pathway of retro-translocationThe retro-translocation pathway can be divided intofour different steps (FIG. 1): substrate recognition andtargeting to the retro-translocation machinery; proteintransport across the ER membrane; release of the sub-strate from the ER membrane into the cytosol; anddegradation of the substrate. However, it is unlikelythat all substrates actually use the same pathway andso, the general mechanism outlined below might differin specific cases.

transmembrane regulator, which cannot easily reachtheir native conformation, as well as proteins, such asthe 3-hydroxy-3-methylglutaryl acetyl-coenzyme-A(HMG CoA) reductase, which are degraded in a regu-lated manner11.

Although misfolding might be the general feature ofall substrates, the ER quality-control system seems unableto deal with heavily aggregated proteins. In fact, morethan 100 years ago, William Russell described a peculiarintracellular structure12 (now called Russell bodies; forreview see REF. 13) that consisted of dilated ER cisternaeand contained aggregated immunoglobulins. Theseaggregates are apparently not subject to degradation.Another example is provided by an artificial fusion pro-tein that contains several dimerization domains thatresult in the formation of aggregates in the ER14. Theseaggregates led to the deformation of the organelle butwere not degraded. Membrane proteins might behavesimilarly. Misfolded influenza haemagglutinin can aggre-gate in the ER, but is not degraded15.Aggregates in the ERmight compromise cell function, but in many casesmight not cause immediate cell death, possibly becausethey do not expose large hydrophobic patches that couldbind chaperones; so they might be similar to INCLUSION

BODIES in the cytosol, which are also relatively benign. Ittherefore seems that the degradation system only attacksproteins that are neither correctly folded nor completelymisfolded and aggregated.

Although we might assume that misfolded ER pro-teins would never leave the ER along the secretory path-way, there is evidence in yeast that cycling between theER and Golgi is necessary for the degradation of severalmisfolded soluble ER proteins. Mutations in compo-nents that are involved in vesicular transport to andfrom the Golgi apparatus retard degradation16,17. Inaddition, a protein component — Bst1 — has been dis-covered in yeast that might be required for vesiculartransport of misfolded, soluble proteins out of the ER.Several misfolded membrane proteins do not seem torequire this detour for their degradation16. Perhaps ERdegradation is confined to certain ER regions that solu-ble proteins reach by vesicular transport through theGolgi compartment and that at least some membraneproteins reach by diffusion in the plane of the ER mem-brane. In support of localized ER protein degradation,

Box 1 | Degradation of proteins by the ubiquitin–proteasome system

Most proteins that are degraded in the cytosol are first polyubiquitylated and then hydrolysed by the proteasome. Theattachment of the polyubiquitin tag occurs in several steps. First, the small protein ubiquitin is activated by attachmentof its carboxy-terminal carboxyl group to a cysteine residue in the activating (E1) enzyme. The E1 enzyme uses thecleavage of ATP into AMP and PP for its activation. Second, ubiquitin is transferred to a cysteine residue of an ubiquitin-conjugating enzyme (E2). Each cell has several E2 enzymes. Third, the ubiquitin is transferred to an amino group of asubstrate, most frequently the ε-amino group of a lysine. This requires E3 enzymes — ubiquitin-ligases — which eithertransiently form a thiolester with ubiquitin, or allow the E2 enzyme to transfer the ubiquitin to the substrate.Polyubiquitylation occurs by attachment of extra ubiquitin molecules to lysine residues in ubiquitins that are alreadyattached. Polyubiquitylated substrates are bound to the proteasome, which is a large particle that consists of twoparticles — a 20S proteolytic and a 19S regulatory particle. The 20S particle is a cylinder with the active sites in itsinterior. The 19S particle has at its base a ring of six ATPases, which are associated with the 20S particle and feed thepolypeptide chain into the interior of the 20S particle so that it is processively degraded. The polyubiquitin chain isremoved by isopeptidases from the substrate before its movement into the proteolytic chamber.


R E V I E W S

CARBOXYPEPTIDASE Y

Carboxypeptidase Y (CPY) is aprotease that is transported fromthe endoplasmic reticulum (ER)to the vacuole of Saccharomycescerevisiae cells. A misfoldedversion, CPY*, is retained in theER, retro-translocated into thecytosol and degraded by theproteasome.

MICROSOMES

A membrane fraction thatconsists largely of ER, andsediments slowly in thecentrifuge.

ER lumen, which is involved in the formation and re-shuffling of disulphide bridges during the folding ofpolypeptides. Its function in ER protein degradationwas initially proposed by experiments in yeast. Cells thatcontained a PDI mutant with a defective peptide-bind-ing domain showed retarded degradation of misfoldedCARBOXYPEPTIDASE Y, and MICROSOMES that were isolated

Substrate recognition. For translocation in the forwarddirection, substrates are selected by virtue of hydropho-bic peptide segments, either signal sequences or trans-membrane (TM) domains, which are recognized in thecytosol by the signal-recognition particle, and in themembrane by the translocation channel1 (BOX 2).Obviously, in retro-translocation, the recognition ofsubstrates must be different, at least for soluble proteins,which lose their signal sequence after import into theER. As misfolded proteins are the main class of sub-strates, the exposure of hydrophobic polypeptidepatches might be a general signal for recognition, andthe ER lumenal chaperones that recognize thesehydrophobic regions might be the signal receptors.However, during the folding of a polypeptide, the samehydrophobic surface patches might be transientlyexposed. How then is the distinction made between aprotein on the folding pathway and one that is destinedfor degradation?

One possibility is that the cell recognizes when a pro-tein exposes hydrophobic surface patches for too long.Such a timing mechanism has been proposed for mis-folded glycoproteins (FIG. 2; for a more detailed review,see REF. 27). Nascent polypeptides that translocate intothe ER lumen are often modified at Asn residues by theattachment of the sugar moiety Glc

3Man

9GlcNAc

2. The

terminal three glucose residues are then sequentiallyremoved by the action of glucosidases I and II. In highereukaryotes, as long as the polypeptide remainsunfolded, a glucose residue is repeatedly added by UDP-glucose glycosyltransferase (UGGT) and cleaved offfrom the sugar chain by glucosidase II. The protein isretained in the ER by its interaction with calnexin andcalreticulin — two related chaperones, which arelocated in the membrane and lumen of the ER, respec-tively, and recognize the monoglucosylated form. Inyeast, the UGGT pathway has not been found, and themisfolded protein might be retained in the ER by a dif-ferent mechanism. However, in all eukaryotes, the timerseems to be provided by the enzyme mannosidase I,which removes the terminal mannose residue, produc-ing the sugar moiety Man

8GlcNAc

2 (REFS 28–30). The

mannosidase reaction is slow29, so only hopelessly mis-folded proteins that spend an excessive amount of timein the ER will be trimmed (FIG. 2). The Man

8-species

seems to be recognized by a lectin, which is related to α-mannosidase but lacks enzymatic activity (called Htm1or Mnl1 in yeast, and EDEM in mammals)31–33. Thelectin might target the protein to the retro-translocationpathway. As expected from the model, inhibition ofmannosidase I or deletion of the Man

8-recognizing

lectin stabilize some misfolded glycoproteins32–35. Whichtiming mechanism targets non-glycosylated proteins isnot known.

Although all chaperones act on folding polypeptides,only a few might be involved in ER protein degradationbecause this probably requires some interaction of thechaperone with the retro-translocation machinery. Sofar, we only have candidates for proteins that mightserve this linker function, the best one being proteindisulphide isomerase (PDI) — an oxidoreductase in the

Figure 1 | Pathway of retro-translocation. In step 1,misfolded lumenal proteins are recognized and unfolded byendoplasmic reticulum (ER) chaperones and targeted to thetranslocation channel. Bacterial toxins probably use the samemechanism for unfolding. Misfolded membrane proteins mightbe targeted to the channel by diffusion in the plane of themembrane (dashed arrow). In step 2, polypeptides are movedthrough the channel. Polyubiquitylation (Ubn) occurs when thepolypeptide chain becomes accessible in the cytosol. In step3, polyubiquitylated polypeptides are released from the ERmembrane into the cytosol. In step 4, polypeptides aredegraded by the proteasome.

Ubn

Ubn

Misfolded substrate Toxins

Chaperone

Unfolding and targeting

ER lumen

Cytosol

Transport through the channel and polyubiquitylation

Release

Degradation

Proteasome

1

2

3

4


R E V I E W S

A role for PDI in retrograde translocation is also pro-posed by recent experiments on cholera toxin41. Theholotoxin is normally very stable, but in the ER lumenthe A1 chain is released from the rest of the toxin, so thatit can be retro-translocated into the cytosol. An extractthat contains ER-lumenal proteins could both disassem-ble the holotoxin and cause unfolding of the A1 chain, asdetected by its increased protease sensitivity. After frac-tionation of the extract, the main unfolding activitycould be attributed to PDI. PDI acts as a new redox-dri-ven chaperone: in its reduced state, it binds and unfoldsthe substrate, and in its oxidized state, it releases it. Anattractive possibility is that the substrate is released fromPDI and directly transferred to the retro-translocationmachinery (BOX 3). The unfolding of cholera toxin in theER lumen indicates that the unfolded state of a polypep-tide chain might be the point at which the toxin and cel-lular quality-control pathways merge (FIG. 1). The‘unfolded state’ might be a relatively subtle alteration inthe conformation of a protein; for example, cholera toxinis only recognized by PDI after its A-chain has beenclipped41, which seems to result in a small local change.

Other chaperones have also been implicated in tar-geting substrates to the degradation machinery. BiP, aheat shock protein 70 family member in the ER lumen,associates with unassembled immunoglobulin lightchains before their degradation42. In yeast, mutants inthe homologue of BiP (Kar2) block the degradation ofseveral soluble substrates43,44, but not the degradation ofmembrane proteins. BiP mutants accumulate ER pro-teins as stable aggregates. A similar effect is seen in dou-ble deletions of two partners of BiP — Scj1 and Jem1

from the mutant were inefficient in the degradation ofmisfolded PRO-α-FACTOR. When retro-translocation wasblocked in sec61 mutants, which disrupt the protein-conducting channel (see below), pro-α-factor wasfound to be associated with PDI36. As this substrate lackscysteine residues, the effect of PDI cannot be explainedby its classical function in disulphide-bridge formationor isomerization. In other cases, however, reduction ofdisulphide bridges by PDI seems to be a prerequisite forretro-translocation37–39. The PDI-related protein Eps1might also have a role in retro-translocation because itsdeletion in yeast prevents the degradation of misfoldedplasma membrane [H+]ATPase40.

Box 2 | Translocation into the endoplasmic reticulum

Polypeptide translocation across the endoplasmic reticulum (ER) membrane can occurco- or post-translationally. The first step in co-translational translocation is targeting tothe translocation channel. When a hydrophobic polypeptide segment, such as a signalsequence, has emerged from the ribosome, it is bound by the signal recognition particle(SRP), and the complex of ribosome, nascent polypeptide, and SRP binds to the SRPreceptor in the ER membrane. The signal sequence is then transferred into the channel,which functions as a second signal-sequence receptor. In post-translationaltranslocation, the signal sequence binds directly to the channel. The translocationchannel is formed from several copies of the Sec61 complex, which is an assembly ofthree membrane proteins. There are homologues of the Sec61 complex in all organisms,including bacteria and archaebacteria. The conserved subunits are the α-subunit, whichspans the membrane ten times, and the γ-subunit, the essential region of whichcomprises a single transmembrane domain. The channel only provides a passiveconduit for polypeptides, and it therefore needs to associate with partners that movesubstrates through it (FIG. 3). Depending on the partner, translocation can occur bydifferent mechanisms.

Figure 2 | Timing mechanism for glycoprotein degradation. The terminal glucose residues are removed from newly synthesizedN-glycosylated endoplasmic reticulum (ER) proteins by the action of glucosidases I and II (dashed line). In higher eukaryotes,misfolded proteins undergo a continuous cycle in which a glucose is attached to the sugar chain by the enzyme UDP-glucoseglycosyltransferase (UGGT) and cleaved off by glucosidase II. The monoglucosylated state is recognized by the chaperones calnexinand calreticulin, which retain the unfolded protein in the ER. The deglucosylated protein can escape the cycle by folding (foldingpathway). In yeast, the retention of misfolded proteins might occur by a different mechanism. In all eukaryotes, mannosidase Icleaves off a mannose residue if a protein stays in the ER for an extended period, and the protein is then targeted to the degradation(disposal) pathway.

Foldingpathway

Glucosylation–deglucosylation cycle

Calreticulin CalnexinSecretorypathway

Foldedglycoprotein

GlucosidaseI and II

Man9GlcNAc2

Glc3Man9GlcNAc2

Man9GlcNAc2 Man8GlcNAc2

Glc1Man9GlcNAc2

Disposalpathway

Mannosidase I LectinDegradation

UGGT Glucosidase II

Unfoldedglycoprotein

PRO-α-FACTOR

Pro-α-factor is the precursor ofα-factor, a peptide secreted by S. cerevisiae cells for mating. Inthe ER lumen, pro-α-factor isgenerated by signal-sequencecleavage from prepro-α-factorand is glycosylated. Whenunglycosylated, it is misfolded,retro-translocated and degradedin the cytosol.

[H+]ATPase

A plasma membrane protein,which uses the energy of ATP topump protons out of the cell.


R E V I E W S

suggests that it needs to be unfolded before retro-translocation can occur (see below). US2 might attract achaperone that unfolds the complex.

Retro-translocation through the Sec61 channel? Aftertargeting to the retro-translocation machinery, apolypeptide needs to cross the ER membrane (FIG. 1,step 2). It is generally believed that this occurs by trans-port through the protein-conducting channel that isformed by the Sec61 complex.

The Sec61 channel and its relative in bacteria(SecYEG) have long been known to be responsible forprotein translocation in the forward direction (forreview, see REF. 1). There are at least three differentmodes in which the channel can function (FIG. 3): co-translational translocation, in which the ribosome is themain partner; post-translational translocation ineukaryotes, in which another membrane protein com-plex, the Sec62–Sec63 complex, and the lumenal proteinBiP are the main partners; and post-translationaltranslocation in bacteria, in which the cytosolic ATPaseSecA is the interacting component. Given its versatility,it is an attractive possibility that the channel — with anappropriate partner — could also function in retro-translocation (FIG. 3, part d).

A role for the Sec61 complex in retro-translocationwas initially proposed by co-immunoprecipitationexperiments20. In cells that express the HCMV proteinsUS2 or US11, MHC class I polypeptides exported fromthe ER could be precipitated with Sec61 antibodies.Although the experiments were carried out under lowstringency conditions (very low salt concentrations), co-precipitation seemed to be specific for substrate mole-cules on their way out to the cytosol. Similar experi-ments showed the association of cellular, misfolded ERdegradation substrates and a toxin with the Sec61 com-plex53–56. Crosslinking of exported pro-α-factor to theSec61 complex was also reported49. The in vitro exportof the A1 chain of cholera toxin from microsomes couldbe inhibited by ribosomes that presumably boundspecifically to the Sec61 channel26.

Genetic experiments also support a role of the Sec61complex in retro-translocation. In various yeast sec61mutants the degradation of several ER substrates isretarded or prevented43,48,49,57. In a genetic screen devisedto specifically isolate sec61 mutants that were defectivein retro-translocation, most mutations mapped to thelumenal regions of the Sec61 complex58.

Although the data indicate that both soluble andmembrane proteins are exported from the ER throughthe Sec61 channel, the evidence presented so far is notentirely conclusive. In addition, some substrates, such asHMG CoA reductase, are only moderately affected bymutations in sec61 (R. Hampton, personal communica-tion). The sec61 mutations have no notable effect on thestability of Ubc6, a tail-anchored ER protein with a car-boxy-terminal TM domain and a cytoplasmic aminoterminus59. Perhaps at least some membrane proteinscan be extracted without a channel, as proposed for theremoval of misfolded bacterial and mitochondrialmembrane proteins60,61.

(REF. 50). It is therefore possible that BiP and its partnerskeep proteins in a non-aggregated state that is compe-tent for degradation, or retrieve misfolded soluble pro-teins from the Golgi, rather than directly target proteinsto the retro-translocation machinery. Another partnerof BiP, the Sec63 protein, which is a component of thetranslocation channel45, has been proposed to releaseBiP from the substrate before its retro-translocationthrough the membrane46. However, Sec63 loads BiPmolecules onto substrates, rather than releases them45,47.Although Sec63 mutations in yeast moderately stabilizesome misfolded proteins43,48–50, this could be an indirecteffect of its role in forward translocation. A role for cal-nexin in ER protein degradation is even more uncertain:mutants in yeast stabilize misfolded pro-α- factor10, buta block in the association of calnexin with glycoproteinsin mammalian cells leads to enhanced degradation,which indicates that calnexin can normally prevent pro-tein degradation35,51.

As mentioned before, certain viral proteins functionby targeting specific substrates to the cellular degrada-tion pathway. The recently solved atomic structure of acomplex between the lumenal domain of the HCMVprotein US2 and the MHC class I molecule explainswhy substrate selection is so specific; the viral proteinbinds to a site that is formed only on folding of theMHC molecule52. However, the size of the complex also

Box 3 | Models for cholera toxin unfolding and retro-translocation

Two models are depicted in which unfolding of cholera toxin by protein disulphideisomerase (PDI) could be coupled with subsequent retro-translocation. In model 1,oxidized PDI — the predominant form in the ER lumen — is reduced by a lumenalreductase anywhere in the ER lumen. Reduced PDI binds and unfolds cholera toxin. Ero1— an oxidase of PDI110,111 — is associated with the translocation channel (pink) andreleases the toxin from PDI directly into the translocation channel. In model 2, thereductase is associated with the channel. Locally generated, reduced PDI unfolds thetoxin, and oxidation by Ero1 (which might not necessarily be localized in this model)releases the toxin from PDI into the channel.

Reductase

Reductase

Reductase Oxidase Oxidase

Oxidase

Oxidase

Oxidase

SS

SS

HS–HS–

Toxin

ER lumen Cytosol

Reductase Reductase

ER lumen Cytosol

PDI

HS–HS– PDI

Oxidation of PDI andsubstrate release

Oxidation of PDI andsubstrate release

Model 1

Model 2


R E V I E W S

APOLIPOPROTEIN

Lipoprotein particles have amonolayer of phospholipids attheir surface, into whichapolipoproteins are embedded.The latter are translocated acrossthe ER membrane in liver cellsand associate with cholesterolesters as they emerge on thelumenal side of the ER.

β2-MICROGLOBULIN

A soluble, lumenal ER protein,which associates with the heavychain of the majorhistocompatibility class Icomplex.

If retro-translocation occurs through the Sec61channel, it seems likely that the substrate needs to be atleast partially unfolded to fit through the pore. Althoughfluorescence quenching experiments indicated that thetranslocation channel has a wide pore of 40–60 Å diam-eter62, electron-microscopy structures of the channelbound to the ribosome show only a narrow opening orno opening at all63,64, and recent experiments indicatethat even small polypeptide domains of 35 Å diametercannot fold inside the channel (M. Kowarik and A.Helenius, personal communication). However, as glyco-proteins are retro-translocated, the channel pore mustbe wide enough to accommodate a polypeptide with anattached carbohydrate chain.

How could a polypeptide chain be brought into thechannel to begin retro-translocation? The simplest pos-sibility is that the polypeptide never fully disengagesfrom the channel after forward translocation. ForAPOLIPOPROTEIN B there is indeed evidence that the proteinremains associated with the channel before it isdegraded65. However, this might be an exceptional casebecause in most other systems completely translocatedproteins66 and toxins can serve as substrates. TheHCMV protein US2 interacts with the folded complexof MHC class I heavy chain and β2-MICROGLOBULIN52, andfolding and assembly of membrane proteins can proba-bly occur only after their complete release from thechannel into the surrounding lipid phase. One thereforehas to invoke mechanisms by which polypeptide chainsare threaded back into the channel pore. In the case of amembrane protein, the TM domain might enter thechannel laterally from the lipid phase, and reverse theprocess of its membrane integration67. The mechanismby which a soluble polypeptide chain would be insertedinto the channel is completely unclear.

Polyubiquitylation of substrates. Many substrates areknown to undergo polyubiquitylation (BOX 1) duringtheir export from the ER membrane into the cytosol(for review, see REF. 11), and this modification is requiredfor degradation. For example, if polyubiquitylation isprevented by the expression of ubiquitin with a muta-tion in Lys48, substrates are stabilized both in yeast68,69

and in mammalian cells70,71. In a temperature-sensitivemutant of the ubiquitin-activating (E1) enzyme, degra-dation is inhibited at the non-permissive tempera-ture54,70–72. Mutations in many components of the ubiq-uitylation machinery stabilize misfolded proteins in theyeast ER (see below). In a permeabilized cell system, thedepletion of ubiquitin from the added cytosol blocks theHCMV protein US11-induced dislocation of MHCclass I heavy chains; re-addition of ubiquitin restoresdislocation and degradation. Mono-ubiquitylation wasinsufficient to trigger dislocation as shown by the re-addition of modified ubiquitin that could not formpolyubiquitin chains73.

ER degradation substrates are polyubiquitylated atthe cytosolic face of the ER membrane, before theircomplete export into the cytosol68,73,74. So, the ubiquity-lation machinery is probably associated with the ERmembrane. Substrates at the ER membrane seem to

Figure 3 | Different modes of translocation. The protein-conducting channel that is formed by the eukaryotic Sec61complex or the bacterial SecYEG complex is a passive porethat can associate with different partners, which results indifferent modes of translocation. The four known modes aredepicted. a | In co-translational translocation, the ribosome isthe main partner. The elongating nascent chain is transferreddirectly from the tunnel in the ribosome into the pore of theSec61 channel. The energy for translocation is derived fromGTP hydrolysis during translation. b | In post-translationaltranslocation in yeast, the tetrameric Sec62–Sec63 complexand the lumenal ATPase BiP are the main partners. BiP servesas a ratcheting molecule to prevent back-sliding of apolypeptide chain through the channel into the cytosol. BiPbinding to the polypeptide chain is mediated by the J-domainof Sec63. ATP hydrolysis by BiP is required for theassociation–dissociation cycle. A similar mechanism of post-translational translocation might occur in othereukaryotes. c | In post-translational translocation in bacteria,the cytosolic ATPase SecA is the interacting component. ATPhydrolysis by SecA is required to push the polypeptide throughthe channel. d | In retro-translocation, an unknown partnermight allow the Sec61 channel to transport proteins in thereverse direction. ER, endoplasmic reticulum.

ER lumen

Cytoplasm

RibosomemRNA

GTP GDP

ATP ADP

ATP ADP

Sec61 complex

ER lumen

Cytoplasm

Periplasm

CytoplasmSecYEG complex

ER lumen

Cytoplasm?

SecA

BiP

a Co-translational

b Post-translational (yeast)

c Post-translational (bacteria)

d Retro-translocation

Sec61 complex

Sec61 complex

Sec62/63 complex


R E V I E W S

AAA FAMILY OF ATPases

ATPases, which are associatedwith diverse cellular activities,contain a conserved domain of200–250 amino acids. Thisdomain includes Walker A and Bmotifs, which are required forATP binding and hydrolysis.

SNARE COMPLEXES

(SNARE, soluble N-ethylmaleimide sensitivefactor attachment proteinreceptor). A vesicle-bound v-SNARE polypeptide associateswith the three chains of a t-SNARE in the targetmembrane to form a stablecomplex, which might drivefusion of the two membranes.

Release of polypeptides into the cytosol. Polyubiquitylationalone seems to be insufficient to release polypeptides fromthe ER membrane into the cytosol.When polyubiquityla-tion is carried out in a permeabilized cell system in thepresence of the ATP analogue AMPPNP, the modifiedchains remain associated with the ER membrane (D.Flierman, C. Shamu,V. Chou and T.A.R., unpublishedobservations), which indicates that another ATP-depen-dent step is required for their release into the cytosol (FIG.1,step 3). One possibility is that the proteasome pullspolyubiquitylated chains out of the membrane93. The19S regulatory particle (BOX 1) can recognize a polyubiq-uitylated substrate, and the six ATPases at the base of theparticle could pull on a polypeptide and thread it intothe adjacent proteolytic chamber. However, inhibition ofthe proteolytic activity of proteasomes can lead to theaccumulation of non-degraded substrates in the cytosol,rather than in the ER20,70,94,95, and ER membranes con-tain only small amounts of bound proteasomes96. Also,mutation of the 19S regulatory particle does not preventretro-translocation of a substrate from the yeast ER91.

Recent experiments indicate a role for a memberof the AAA FAMILY OF ATPases — Cdc48 in yeast and p97in mammals — in extracting polypeptides from theER membrane75,91,97–99. Cdc48/p97 is closely relatedto N-ethylmaleimide-sensitive fusion protein (NSF), anATPase that disassembles SNARE COMPLEXES after vesiclefusion. The structure of these ATPases indicates thatthere are two hexameric rings stacked on each otherwith a pore in the middle100. Cdc48/p97 was known tofunction in the homotypic fusion of ER membranesand in the assembly of the Golgi (for review, see REF. 101).In these reactions it acts in a complex with the cofactorp47 (REF. 102).

Several lines of evidence now indicate thatCdc48/p97 functions with two other cofactors, Ufd1 andNpl4, in the ER-protein-degradation pathway. Theseproteins form a stable complex in both yeast and mam-mals103–105. In yeast mutants of cdc48, ufd1 or npl4, thedegradation of several misfolded ER substrates isretarded75,91,97–99. Most misfolded proteins accumulateinside the ER, which results in the induction of the UPRand leads to the compensatory upregulation of ER chap-erones75,98. Polyubiquitylated polypeptide chains accu-mulate at the membrane in npl4 and ufd1 mutants91,97,which indicates that this modification precedes the stepthat involves the Cdc48–Npl4–Ufd1 complex.

The mammalian homologue of Cdc48 — p97 —might have a similar role in ER protein degradation. Inpermeabilized mammalian cells that express the HCMVprotein US11, the addition of a dominant-negative p97protein that is deficient in ATP hydrolysis blocks the dis-location of MHC class I heavy chains from the ER mem-brane into the cytosol75. A similar effect was seen onaddition of p47, presumably because this cofactordiverted p97 away from its function in ER degradation.In both cases, polyubiquitylated MHC class I heavychains accumulated at the ER membrane. When wild-type p97 protein was added to permeabilized cells andthe membranes were subsequently solubilized in a milddetergent, both unmodified and ubiquitylated MHC

carry somewhat shorter polyubiquitin chains than thosein the cytosol75, which indicates that a certain chainlength is required for release from the membrane.

The components of the ubiquitylation system thatare involved in ER degradation have been identified inyeast, but there are also homologues in mammals. Asexpected, many of the components are bound to the ERmembrane. The relevant ubiquitin-conjugatingenzymes (BOX 1) (E2 enzymes or Ubcs) include Ubc7,which is anchored to the membrane through its partnerCue1 (REF. 76), and Ubc1 (REF. 5). Ubc6, an ER-membraneprotein77, also has a role. Mutations in these enzymeslead to the stabilization of various substrates68,69,76,78–80.An E3 ubiquitin-ligase, Hrd1/Der3, which is specificallyinvolved in ER-protein degradation, has been identifiedin two independent genetic screens81–83. Hrd1/Der3 is amulti-spanning ER membrane protein with a carboxy-terminal RING-H2 domain facing the cytosol, whichbinds the conjugating enzymes Ubc1 or Ubc7 (REFS

84,85). The RING-H2 motif is characteristic of a group ofubiquitin ligases. Hrd1/Der3 is stabilized by its partnerprotein in the ER membrane, Hrd3 (REFS 86,87). OtherER-associated ubiquitin ligases have been identified andhave a role in the degradation of certain substrates88,89.

Whether polyubiquitylation is a general feature ofretro-translocation is unclear. Some toxins exportedfrom the ER into the cytosol lack lysine residues, butpolyubiquitylation might occur at the aminoterminus90, as suggested for a mutant T-cell receptorsubunit that lacked lysine residues and yet was still ubiq-uitylated and degraded71. However, the degradation ofmisfolded pro-α-factor does not depend on theHrd/Der proteins, and probably does not even dependon ubiquitylation (REF. 11).

The precise role of polyubiquitylation in retro-translocation remains to be clarified. Because substratesaccumulate inside the ER when polyubiquitylation is pre-vented54,72,76,91, it seems possible that the modification isrequired for retro-translocation per se, and not just fordegradation by the proteasome. However, at least partialtranslocation of the polypeptide must occur before thecytosolic modification reaction can occur. This is eventrue for membrane proteins with cytosolic domains:removal of all lysines from the cytosolic tail of the MHCclass I heavy chain did not prevent its US11-mediated dis-location or its polyubiquitylation74, which indicates thatthe lumenal domain must have moved into the cytosolbefore polyubiquitylation occurred. Once the substrate isaccessible, polyubiquitylation could serve as a ratchetingmechanism to move extra polypeptide segments into thecytosol; the bulky modification at lysine residues couldprevent these segments from moving back through thechannel into the ER lumen92. If this occurs, it might notbe the only mechanism for moving substrates becausepolyubiquitylation seems to be insufficient to transportlumenal substrates completely into the cytosol.Another,not mutually exclusive, possibility is that a polyubiquitinchain serves as a recognition signal for downstream com-ponents, specifically the proteasome or the Cdc48/p97complex (see below). These could use ATPase activity toactively move the polypeptide chain.


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a tail-anchored transcription-factor precursor from theyeast ER membrane into the cytosol104.

The role of the Cdc48/p97 complex might be similarto that proposed for AAA proteases in bacteria andmitochondria (FIG. 4). These proteins contain an amino-terminal TM domain, followed by the AAA ATPase seg-ment and a protease domain (reviewed in REF. 108). BothFtsH in the inner membrane of Escherichia coli, andYta10/Yta12 and Yme1 that face the two sides of the innermembrane of mitochondria, have been shown to bind amisfolded membrane protein at one end, and to proces-sively pull it out of the membrane for degradation60,61.

How exactly the Cdc48/p97–Ufd1–Npl4 complexrecognizes substrates is unclear. Some data indicate thatCdc48/p97 interacts directly with ubiquitin104,109,whereas other data indicate that it can interact withnon-ubiquitylated polypeptides75. It also remains to beclarified whether the Cdc48/p97 complex could haveany further role in polyubiquitylation (for example, inchain extension), and whether it can deliver substratesdirectly to the proteasome.

PerspectiveOur knowledge of retro-translocation is still rather lim-ited. We have presented here a general pathway, but it islikely that this pathway does not apply to all ER sub-strates. In fact, a list of required components for yeastsubstrates shows many differences11. The componentsthat are implicated in retro-translocation need to bestudied in much more detail. For example, is there anassociation of PDI-interacting components with theSec61 channel? Is the Hrd1/Der3 ubiquitin ligase associ-ated with other membrane proteins? How is Cdc48/p97bound to the ER membrane? Another important ques-tion in the field is where exactly retro-translocationtakes place. Is it restricted to certain domains of the ER.Most studies so far have been carried out with intactcells, which have allowed little mechanistic insight.Progress will depend on the establishment of in vitrosystems that recapitulate the process, and also on thegeneration of defined translocation intermediates,which have proven so useful in the study of forwardtranslocation. The final goal must be a reconstituted sys-tem that consists of purified components. Clearly, thefield of retro-translocation is still in its infancy andpromises to be an exciting area for many years to come.

class I heavy chains associated with p97. The associationwas dependent on US11 and was specific for membrane-associated heavy chains, but it is not yet clear whetherp97 directly binds to the substrate. Mutant p97 that isdefective in ATP hydrolysis also associated with heavychains, which indicates that p97 requires ATP hydrolysisto release polyubiquitylated molecules into the cytosol.One mechanism by which this might occur is that thehexameric ring of Cdc48/p97 binds to the ER membraneand pulls the polypeptide chain, perhaps by moving itthrough the central pore. This would be analogous to themechanism by which ring-shaped hexameric helicasesmove along single-stranded nucleic acids106, or by whichhexameric ATPase rings move polypeptides into the pro-teolytic chambers of the eukaryotic proteasome or thebacterial ClpP protein107. Aiding the movement of apolypeptide chain might also be the way in which thecomplex of Cdc48, Ufd1 and Npl4 releases a fragment of

Figure 4 | Extraction of proteins from membranes by AAA ATPases. a | The bacterial AAAprotease FtsH and the mitochondial inner membrane AAA proteases Yta10/Yta12 and Yme1 pullunfolded membrane proteins out of the membrane and degrade them. These proteins contain atransmembrane segment (green), an ATPase domain (purple), and a metalloprotease domain(grey). It is possible that the polypeptide moves through the central pore of the ATPase ring. b | The AAA ATPase Cdc48 (purple) and its partners Ufd1 and Npl4 (yellow and blue,respectively) release unfolded, polyubiquitylated (Ubn) polypeptides from the endoplasmicreticulum (ER) membrane into the cytosol. The complex might be associated with the ERmembrane through an unknown protein (shown in green) and might accept polypeptides thatemerge from the translocation channel (grey).

Substrate

ATP

ADP

AAA protease

Substratepulling/degradation

Substrate

ATP

ADP

Cdc48/Ufd1/Npl4 complex

Substratepulling/releasing

ER lumen

Cytosol Ubn

a AAA protease b Cdc48/Ufd1/Npl4

ClpP

A bacterial protease, which, likethe eukaryotic proteasome,forms a cylinder with active sitesin the interior. A ring of ATPasesthat is formed by ClpA feedpolypeptides into ClpP.

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AcknowledgementsWe thank T. Sommer, K. Matlack, H. Ploegh and D. Finley for criti-cal reading of the manuscript and helpful comments. B.T. is a fellowof the Damon Runyon Cancer Research Foundation and Y.Y. is aHelen Hay Whitney Fellow. The work in the authors’ laboratory wassupported by the National Institute of Health. T.A.R. is a HowardHughes Medical Institute Investigator.

Online links

DATABASESThe following terms in this article are linked online to:Interpro: http://www.ebi.ac.uk/interpro/RING-H2 domainLocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkPDISaccharomyces Genome Database: http://genome-www.stanford.edu/Saccharomyces/Cdc48 | Cue1 | Eps1 | Ero1 | Hrd1 | Hrd3 | Htm1 | Jem1 | Kar2 |Npl4 | Scj1 | Sec61 | Sec62 | Sec63 | Ubc1 | Ubc6 | Ubc7 | Ufd1 |Yme1 | Yta10 | Yta12Swiss-Prot: http://www.expasy.ch/Bst1 | cystic fibrosis transmembrane regulator | E1 enzyme | EDEM |HMG CoA reductase | NSF | p97 | UGGT | US2 | US11Access to this interactive links box is free online.

retro-translocation of proteins from the endoplasmic reticulum into the cytosol

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