Protein targeting to and translocation across the membrane of the endoplasmic reticulum
Jodi Nunnari and Peter Walter
University of California, San Francisco, California, USA
Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum. Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae. Results obtained in each of these systems have contributed novel, mostly non-overlapping information. Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon. Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S. cerevisiae, by selecting for mutants in the translocation pathway. None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems. In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.
Current Opinion in Cell Biology 1992, 4:573-580
Introduction tein complex, the SRP receptor (SR, comprised SRa and SRP subunits). Once SRP interacts with its receptor, the In eukaryotic cells, the first step in the biogenesis of pro- signal sequence dissociates from SRP and elongation ar- teins destined to be secreted and IumenaI proteins that rest is relezed. LIpon release from SRP, the nascent chain are residents of the secretory pathway is the targeting ancl inserts into and becomes tightly associated with the ER translocation of these proteins across the membrane of membrane via interactions with components of the ma- the endoplasmic reticulum (ERI. The ER is also the site chinery that mediate the translocation of the polypeptide for the integration of membrane proteins that comprise across the membrane, collectively referred to as the trans- the plasma membrane and other intracellular membranes locon. of the secretory and endocytic pathways. These proteins Following nascent chain insertion, translocation of the are initially synthesized on ribosomes in the cytosol of the nascent chain proceeds through a protein conducting cell and are selectively targeted to the ER. Targeting to the channel across the ER membrane and into the lumen. ER is specified by a signal sequence contained within the Interaction of nascent proteins with BiP, a member of polypeptide chain, usually found at the amino terminus of the protein. the heat-shock protein 70 family that resides in the lu- men of the ER, facilitates the native folding and assem- In higher eukaryotes, the vast majority of proteins are tar- bly of proteins. During co-translational translocation of geted to the ER in a obligatory cotrdnslational, ribosome- nascent polypeptides, enzymes present in the membrane dependent manner. A cytoplasmic ribonucleoprotein, and lumen of the ER modify the polypeptide chain. ER- termed signal recognition particle ( SRP), binds to the sig- specific signaI sequences are cleaved by signal peptidase, nal sequence as it emerges from the ribosome causing an oligosaccharides are covalently attached to the nascent arrest or pause in the elongation of the nascent polypep- chain by oligosaccharyl transferase and disulfide iso- tide. This pause may extend the time in which the nascent merase catalyzes disulfide bond formation. These mod- chain can be productively targeted to the ER membrane. ifications to the polypeptide chain are important for the Targeting of the ribosome-nascent chain-SRP complex proper folding of the protein and the enzymes that cat- to the ER membrane is mediated by the specific interac- alyze these modifications may possibly be involved in the tion of SRP with the ER membrane heterodimeric pro- process of nascent chain translocation.
Abbreviations ER--endoplasmic reticulum; SR-signal recognition particle receptor; SRP-signal recognition particle: SSR-signal sequence receptor; TRAM-translocating-chain-associating membrane protein.
@ Current Biology Ltd ISSN 0955-0674 573 574 Membranes
This review encompasses the recent advances made in of the G-domain inhibits binding of the signal sequence understanding the mechanisms and components respon- to the M-domain, which can be reversed by proteolytic sible for the specific targeting and ttanslocation of pro- removal of the alkylated G-domain. These findings also teins across the ER membrane. suggest that, although the GTPase domain does not bind signal sequences, it may modulate the binding of signal sequences to the M-domain. This prediction is consistent with the fact that GTP is required in the targeting and translocation pathway. Signal recognition, targeting and insertion of pre-proteins into the ER membrane To date, three components involved in protein translo- cation, SRP54, SRa and SRP, are members of the GT- Mammalian SRP is composed of six polypeptides (72, Pase superfamily and have been shown to bind GTP 68, 54, 19, 14 and 9kD) and a 7SL RNA molecule. SRP ([ 10,11,16,17]; J Miller, P Walter, unpublished data). mediates three distinct functional activities: signal recog- SRP54 and SRa form a unique subfamily of GTPases. nition, elongation arrest and translocation promotion [I]. The sequence similarities between SRP54 and SRa sug- These activities are contained within three separate struc- gest that these proteins were derived from a common tural domains of the particle. Elongation arrest requires ancestor [ lo,11 1. SRP is not closely related to other GT- the presence of the 9 and 14kD proteins, which foml Pases by sequence and is also unique among GTPases as a heterodimer that binds to the Alu domain of 7SL it contains an amino-temlinal transmembrane domain (l RNA [2*]. The 68 and 72 kD proteins also form a het- hIiller, P Walter, unpublished data). erodimeric RNA-binding complex and are necessary for Following the targeting of the ribosome-nascent chain- the interaction of SRP with the ER membrane and the SR SRP complex to the ER membrane, the signal sequence [ 2*]. Investigation of the mechanism of assembly of SRP dissociates from SRP, and the nascent chain inserts into in vitro has shown that the binding of the 9/14 kD and and becomes tightly associated with the ER membrane the 68/72 kD heterodimers to 7SL RNA is non-cooperative via interactions with components of the translocon [ 181. in the absence of the 54 and 19 kD subunits of SRP [3-l. In this step, GTP is required for the release of the The 19kD protein binds to 7SL RNA and is required for nascent chain from SRP [ 191. The non-hydrolyzable ana- the binding of the 54 kD subunit (SRP54) to SRP [4,5-l. log, Gpp(NH)p, promotes the release of the nascent SRP54 probably binds directly to a region in 7SL RNA that chain from SRP and its insertion into the ER mem- has been identihed as a phylogenetically conserved motif brane, but prevents the subsequent release of SRP from characteristic of SRP RNAs [6]. Photocrosslinking stud- the SR [ 20**]. Thus, GTP hydrolysis is required for re- ies have demonstrated that SRP54 specifically binds to cycling of both SRP and SR for subsequent rounds of the signal sequence of secretoty proteins and signal-an- targeting and nascent chain insertion. Mutations in the chor sequences in nascent integral membrane proteins GTP-binding consensus sequences of SRa reduce the ef- ]7,8,9*]. ficiency of GTP-dependent nascent chain insertion and Insight into the mechanism of signal sequence recog prevent the formation of a stable SRP-SR complex in nition was gained when the gene encoding SRP54 was the presence of Gpp(NH)p [21-l. These observations cloned [ 10,111. From the deduced amino acid sequence, indicate that the GTPase activity of SRa plays a role in SRP54 is predicted to contain three domains: an amino- targeting and translocation. The specific contribution of terminal domain of unknown function, followed by each of the three GTPases, SRP54, SRa and SRP, in tar- a GTPase domain, which contains the consensus se- geting and nascent chain insertion in the ER membrane, quence motifs for GTP binding. These two domains however, remains to be determined. In general, GTPases will be collectively referred to as the G-domain. SRP54 function to assemble macromolecular complexes in tem- also contains a methionine-rich domain, ternled the M- poral succession. Thus, one might envision that these GT- domain. The M-domain is proposed to contain a signal- Pases function to assemble accurately components of the sequence-binding pocket lined with methionine residues translocon, so that the signal sequence can be specifically that accommodates diverse signal sequences because inserted into the ER membrane [22*]. of their flexibility [ 111. Limited proteolysis confirmed From in zWo studies, the mammalian system has re- the domain boundary between the G- and M-domains vealed great insight into the mechanism of SRPdepen- [ 121. Photocrosslinking studies demonstrated that the dent signal recognition and targeting. It is very likely, signal sequence binding site of SRP54 is contained in however, that other pathways for ER targeting exist. In the M-domain which, in addition, contains an RNA-bind- S. ceretlisine, post-translational ER targeting and trans- ing site [ 12-141. As a free protein, SRP54 can bind signal location have been observed both in llitro and in uivo sequences [ 15**]. Biochemical dissection of free SRP54 [ 23-251. In yeast, other cytosolic factors, the hsp 70s as- demonstrated that the M-domain of SRP54 alone is suit?- sociate with pre-proteins and facilitate post-translational cient to recognize and bind signal sequences. Upon cell translocation [ 26,271. The presence of an SRPindepen- fractionation, all SRP54 is found complexed in SRP (D dent post-translational targeting and translocation path- Zopf and PW, unpublished data). Thus, it is unlikely that way has been demonstrated in l&-o for a small set of free SRP54 plays any role in targeting or translocation. substrates in the mammalian system as well (281. Experimental evidence suggests that the M- and G- The genes encoding the S. cerevisiue homologs of SRP54 domains of SRP54 physically interact [ 15**]. Alkylation and SRa were recently cloned [ 29,30,31**]. Evaluation of Protein translocation apparatus in the endoplasmic reticulum Nunnari and Walter 575 the in vivo role of the SRI’-dependent targeting pathway Translocation of proteins across the was facilitated by the molecular genetics techniques avail- endoplasmic reticulum membrane able in S. cerevisiue. Deletion of the genes encoding ei- ther SRI’54 or SRa, or both, results in viable, but poorly Subsequent to the targeting of a nascent protein, the growing cells, suggesting that the SRE-dependent path- ribosome-nascent chain complex associates with the ER way can be partially by-passed in vivo [31**,32**]. Upon membrane and translocatlon of the nascent chain across depletion of SRP54 or SRa in yeast cells, precursors to the membrane proceeds. It has long been proposed that both secretory and membrane proteins accumulate in protein translocation occurs through a proteinaceous the cytosol [31°0,32**,33*]. The degree to which differ- channel. It has been shown that large ion conducting ent proteins are affected, however, vanes greatly. The channels are present in ER membranes [39,40**]. Con- transkxation of carboxypeptidase Y, a vacuolar pro- ductance through these channels is dependent on the tein, for example, is unaffected in SRI’-depleted cells, release of nascent chains from nbosome-nascent chain whereas the translocation of Kar2p, a lumenal ER protein, complexes engaged in the process of translocation, sug- and dipeptidyl aminopeptidase B, a vacuolar membrane gesting that translocation proceeds through these chan- protein, are severely diminished. Cytosolic precursor to nels [40**]. These findings also suggest that the ribosome Kar2p accumulates in SRP-depleted cells, but a portion of may play a role in keeping the channels open during pro- newly synthesized KaRp is still translocated. As accumu- tein translocation. lated Kar2p precursor cannot be translocated post-trans- lationally, pre-Kar2p is probably targeted co-translation- The protein-conducting channel is likely to be a dy- ally to the ER membrane in an SRI’-independent man- namic structure. Its subunit composition may vary at dif- ner. Thus, there appear to be several possible pathways ferent sequential translocation stages, such as initiation to the ER membrane: an SRI-dependent co-translational of translocation, steady-state translocation and termina- pathway, a post-translational pathway and a possible SRP- tion of translocation. Different pre-proteins may require independent co-translational pathway. It is likely that in the function of different translocation components. This wild-type cells the bulk of protein targeting occurs via may result from the specific targeting pathway that they the SRP-dependent pathway, and that alternative routes utilize, or from topogenic determinants, e.g. stop trans- provide a scavenger pathway only in SRP or SR-deficient fer sequences. One major goal in the field is to identify, cells. Future research will focus on the molecular nature biochemically isolate and determine the function of the of the alternative pathways. It will be interesting to dis- components that play a role in the process of nascent cover whether the pathways utilize the same translocon chain translocation and membrane protein integration. that is used for SRI-dependent translocation. A major advance in the study of protein translocation was the development of a reconstitution method by which Examining the i?l rjirlo role of SRP revealed that pre- translocation competent vesicles can be prepared from proteins can utilize alternative targeting pathways with a detergent extract of ER membranes [41]. With this varying efficiencies, This may explain why previous ge- assay, membrane components involved in translocation netic screens in S. cerezMze failed to detect SRP. A can be identified directly. This reconstitution system has new selection has been used to isolate a translocation- been utilized successfully to fractionate detergent-solubi- defective mutant in a novel gene, SecG5 [ 34**]. This gene lized ER membrane components required for transloca- encodes a homolog to the 19kD subunit of mammalian tion [42*]. It has also been utilized to analyze whether SRR [35**,36**]. The translocation defect present in cells components, identified by other approaches, contribute harboring the mutant allele, se&- 1, confirms the role of to the translocation process [43**]. Irnmunodepletion of SR from the detergent extract, for example, results in a SRP in targeting and translocation in lklo. Biochemical complete loss of translocation activity. This in vitro assay, and genetic studies demonstrate that Sec65p. SRP54p however, may not readily reveal components required for and a small cytoplasmic RNA, scR1, are part of a 16s the regulation of translocation or components that are ribonucleoprotein particle. Sec65p is required for the not rate-limiting for translocation. integrity of the yeast SRP and promotes, as in the case of mammalian SRP, the binding of SRI’54 [36*g]. Crosslinking of nascent chains to membrane compo- nents has been performed to identify components po- tentially involved in translocation. Two groups inde- The in zko role of SRP has also been studied in other pendently identified a 35-39kD ER glycoprotein by eukaryotic organisms. Mutations in the gene encoding an photocrosslinking, and termed the crosslinked product SRP-RNA of Yarrou~ia lzpofyticu exhibit a temperature- signal sequence receptor (SSRa) and mp39, respectively dependent growth phenotype [37*,38-l. At non-pennis- [44,45]. The 35.39kD glycoprotein does not, however, sive temperatures, the synthesis of a major secreted pro- appear to be a signal sequence receptor, because mature tein, alkaline extracellular protease, is dramatically re- portions of secretory proteins can also be crosslinked duced, whereas overall protein synthesis is unatfected. to it [45]. Using the deduced size of the glycopro- This observation suggests that the mutated SRP is de- tein crosslinking target, a polypeptide was puriIied and ficient in membrane targeting, but still functions in its designated the SSRa protein [46]. This polypeptide is ability to arrest translation of pre-proteins. part of a heterotetrameric membrane protein complex 576 Membranes
[43**]. Antibodies raised against SSRcl immunoprecipi- ending the search for the function of ribophotins. Other tate crosslinked nascent chains, and Fab fragments block ribosome receptor candidates, a 180 kD rough ER mem- translocation, consistent with the idea that SSR is close to brane protein and a 35 kD membrane protein, have been the site of translocation [46,47]. To test directly whether identified [ 57,58*]. Ribosome-binding activity solubilized SSR is required for translocation, detergent-solubilized and reconstituted from ER membranes, however, does extracts were immunodepleted of the complex and SSR- not cofractionate with the 180 kD protein, indicating that depleted extracts were reconstituted into artificial vesicles another, as yet unidentified, protein(s) may function as [43**]. Depletion of SSRdoes not affect nascent chain tar- the ribosome receptor [ 590,60*]. Additional experimen- geting, secretory protein translocation or membrane pro- tal evidence suggests that the 180kD protein may not tein integration [43**]. A number of explanations could be required for it, ritro translocation [6I*]. A defnitive account for this apparent discrepancy. SSRcould function demonstration of a ribosome receptor in the future re- in translocation in a manner that is not detected by the quires the candidate protein to bind stoichiometrically to in z&-o translocation assay. Alternatively, SSR may not be ribosomes. required for translocation and may only fortuitously be found in proximity to nascent chains. It is certain, how- In S. cerezhk7e, mutations that disrupt translocation of ever, that the polypeptide identified as SSRdoes not func- pre-proteins across the ER membrane were selected tion as a signal sequence receptor, as its name implies, using pre-protein-enqme fusions. Mutations in three and that it is not required for an essential rate-limiting genes, SEC61, SEC62 and SE@<. Lvhich encode ER step in translocation. membrane-spanning proteins, impair protein transloca- tion [25, 621. It is unclear at the present time whether Further investigation revealed that translocating nascent all pre-proteins require the products of these genes chains can be crosslinked to another glycoprotein in for translocation ill rdllo. Recently. a new mutant al- the same molecular weight range as SSRcl (48**]. Using lele, .sec61-.?, has been isolated and appears to affect crosslinking and reconstitution approaches, the crosslink the translocation of a wide spectrum of secretory pro- target, a membrane glycoprotein termed the translocating teins as well as the integration of membrane proteins chain associating membrane protein (TRAM), was puti- [3-t**). Mutations in Sec62p and Sec6.?p, however, onl! lied. The deduced amino acid sequence indicates that appear to affect the translocation a subset of pre-proteins TRAM is a multispanning membrane protein. In a recon- [ 251. Immiinoprecipitatio~~ and crosslinking experiments stitution assay, TRAM is either stimulatory or required for indicate that Sec6l p. Sec62p and Sec63p are present in a the efficient translocation of several secretory substrates. multisubunit complex with two other proteins of molec- Other ER proteins in the vicinity of translocating nascent ular weights 31.5 and 23 kD , respectively [63]. The yeast secretory and membrane proteins have been identified mgene, which encodes a homolog of the mammalian by crosslinking [9*,49=,50**]. A 34 kD non-glycos)lated RIP, is also necessary for translocation [b~f, 65*]. Mam membrane protein that is distinct from SSRa!and TRAh4 malian BiP. however, does not appear to be required for crosslinks to both nascent secretory and membrane pro- translocation iu rdtro [ 66,671. tein polypeptides [ 49’1. Similarly, several glycosylated and non-glycosylated ER membrane proteins, which are Preproteins in the process of translocation can be in close proximity to membrane proteins containing crosslinked to Sec61p and Kar2p [68**,69**]. Crosslink- stop-transfer or signal-anchor sequences, have been iden- ing of pre-proteins to SecbIp is dependent on functional tified [9’,50**]. Some of these crosslinks may be specific Sec62p and Sec63p [69**], With short nascent chains, to nascent membrane-spanning proteins and, thus, may crosslinks to Sec62p are also observed [6x**], These function solely in the integration of membrane proteins obseniations suggest that Sec62p/Sec63p may act prior (see High and Dobberstein, this issue, pp 581-586). Syr- to Secblp. Although km-2 mutants exhibit translocation thetic signal peptides have also been photocrosslinked defects i)z zdtro, they do not inhibit crosslinking of pre- to specific integral ER membrane proteins [51*]. Much protein to Sec6Ip as severely [690*]. Thus, KaRp may effort in the years to come will be directed at purifying act after Sec6lp in translocation. Crosslinking of nascent these proteins identified by crosslinking and determining chains to SecGlp requires ATP hydrolysis (68**.69**]. The their roles in protein translocation and integration. translocation factor responsible for the ATP-dependent interaction is unknown. Evidence from the mammalian Ribosome-binding sites present in the ER membrane system also suggests that a membrane ATPase is required are thought to be involved in steady state translocation for translocation 170.. 61*]. of nascent chains. Initially, ribophorin I and II were thought to mediate ribosome binding to the ER, but In yeast, additional mutations, termed sec70, sec71 and were subsequently shown not to be involved [52, 53, sec7-? have been isolated and shown to cause defects in 541. Ribophorin I antibodies, however, block protein protein translocation and membrane protein integration translocation, consistent with their being in close prox- [71**]. Future work will focus on cloning these genes imity to ribosomes and translocation sites [ 551. Recently, and determining their role in the process of transloca- it was observed that a membrane protein complex com- tion and membrane integration. The selection for genes prised of both ribophorin I and II and a 48 kD protein is involved in targeting and translocation has not been ex- associated with oligosaccharyltransferase activity [ 56**]. haustive. Thus, it is likely that the development of new se- This suggests that the ribophorins are required to cat- lection schemes for mutations will yield additional genes alyze the attachment of oligosaccharides to proteins, thus involved in the process. Protein translocation apparatus in the endoplasmic reticulum Nunnari and Walter 577
Regulation of protein translocation Acknowledgements
Translocation of pre-proteins across the ER membrane JN is supported by a Gordon Tomkins Fellowship from UCSF. PW is modukdted in several ways. Determinants contained is supported by grznts from Alfred P Sloan Foundation and NIH. within the protein, such as stop-transfer sequences, must signal the translocation apparatus via some mechanism. Recently, another topogenic determinant has been dis- References and recommended reading covered [ 72**] : signals contained within apolipoprotein B can mediate a pause in translocation. Whether a spe- Papers of particular interest, published within the annual period of re- cific translocon component mediates this pdux in trans- \ic\v. have been highlighted as: location remains to be determined. . of special interest . . of outstlmding interesI There is increasing evidence that the ribosome also plays I. SIIXXI. V. WU.IXH I’: Each of the Activities of Signal Recogni- tion Particle (SRP) is Contained within a Distinct Domain: a role in the regulation of translocation. Ionic conduc- Analysis of Biochemical Mutants of SRP. Cell 1988, 52:39+9. Lince through the putative protein translocation chan- 2 SIX~lI~ K;. hlos\ J, U’,u:reH I’: Binding Sites for the 9.Kilodal- nels in the ER membrane depends on the presence of . ton and Il-kilodalton Heterodirneric Protein Subunit of the a ribosome engaged in translocation of a nascent chain Signal Recognition Particle (SRP) Are Contained Exclusively (-iO**]. Consistent with these findings, it was shown l-q in the Alu Domain of SRP-RNA and Contain a Sequence cx-osslinking that membrane proteins in the process of Motif that is Conserved in Evolution. ,Ilol Cell Viol 1991, 1 1:39i~~3959. integration remain in the vicinihz of specific ER proteins The inlrraction of SRP9 and SRPlt with SW-RNA is located to four until termination of translation occurs [SO-] Upon ter- regions !\\ithin ;i specihc domain related to the Alu family of repetitive mination, crosslinks to these ER proteins no longer form. DNA sequencc.s. One of these region5 is consewed in evolutionarily Even after the cyttoplasmic tail of a nascent membrane diverse SKPKNAs. sumesting that SIUY 1-1 homologs may exisr in these c)rganism.s. protein has been lengthened I-q, nearl~~100 amino acids. 3 J:L’zL~ F, W’AIU(~H I’. JOHNSON AE: Florescence-Detected As- the stop-transfer signal remains in the viciniv of spccifc . sembly of the Signal Recognition Particle: the Binding of ER membrane proteins. This suggests that the ribosomc, rhe Two SRP Protein Heterodimers fo SRP RNA Is Non- upon termination of translation, transduces a signal to cooperative. Rio&~,r&:cl,?~ 1992. in press. the translocon to coniplctc membrane protein integra- Fluorcsence specrroscopy \XXS used to esamine the assembly of SRP b) attaching a tlourorscrin to SRFKNA. The hererodimers SRP68 ‘72 and tion [50-l. SRI’9 1-1 bmd Io the RNA in a non-coopentive nlannrr in the absence of SKI’19 and SKI%. 4. Srec,~r~. \‘. Vi’.xmitx I’. Binding Sites of the I9-kDa and 68/72- kDa Signal Recognition Panicle (SRP) Proteins on SRP RNA as Determined by Protein-RNA ‘Footprinting’. PTDL‘ N&l .-lcttd sci 1’SA IO%+ 85: 1X01-1805. Conclusion i. Z\!wi% C: Interaction of Protein SRP19 with Signal Recogni- . tion Particle RNA Lacking Individual RNA-helices. Nftc~ek The mechanisms employed for targeting and trdnslmx Acids KKS 1991, 19:2955-2960. tion of pre-proteins across the ER membrane have onI), Thr binding site of SKP19 to SRI-RNA was examined by mutageniz- begun to be resolved. Three distinct GTPases are known ing SKI’-RNA. SKPl9 hinds mamh to helix 6 of SRP-KNA, hut also requires elemenrs from the rvol&narily conserved pan of SRP.Rh’A to interact during protein targeting, and the functional These resuks indicate rhat helix 6 is close to the evolutionarily con- importance of the individual GTP-binding sites is still sened domams of SRP-KNA and sugResr a model for the a%embly of a mysteq. In addition to SRPmediated targeting, there SRI’. appear to be other targeting pathways to the ER. Future 6. PoKrrz MA. SI’RI’I~ K, W.UU(I+R I? Human SRP RNA and E. coli goals will he to ident@ components in other targeting 4.5s RNA Contain a Highly Homologous Structural Domain. pathways and to determine their relati\re importance in Cdl 1988, 55:+-6. pry-protein targeting i,r ldrv. KI’K%CI IAUA n’. Wmn~wN M. GIR+IOVICH AS, BOCHUIUZVA ES, BIEI.U 1-l. ~OPORT TA: The Signal Sequence of Nascent Pre- prolactin Interacts with the 54K Polypeptide of the Signal A number of putative components of the trdnslocon have Recognition Particle. Nftlfire 1986. 320:63-t-636. been identilied. Surprisingl~~ however, at present there Kt~ec; 1:C. W.UEH I’. JOHNSON AE: Photocrosslinking of the is no correspondence between the components identi- Signal Sequence of Nascent Preprolactin to the 54-kilodal- tied in yeast and mammalian cells. Much of the effort in ton Polypeptide of the Signal Recognition Particle. Proc the lield will be devoted to obtain additional membrane Ncitl ~ccrd Sci 1 ‘SA 1986. 83:860+8608. components and to decipher their mechanistic function HIGH 5. GOfu~cn D, Wi~:.rmwN M. Rwol~owr TA, DOBHEICXEIN 8: The identification of Proteins in the Proximity of Signal- in translocation. In pursuing this goal, we will gain insight anchor Sequences during Their Targeting to and Insertion into whether diRerent pre-proteins may require a differ- into the Membrane of the ER. .I Cell Biol 1991, 1153544. ent subset of components for translocation as a result of Signal anchor domains of membrane proteins crosslink to SRPSq. Sev the specific targeting pathway that the)! use or as a re- cral novel ER membrane proteins were also shown to photo-crosshnk sult of specific topogenic determinants contained within to membrane proteins during the process of integration. them. Insights wivill also be gained into the regulator) 10. ROLIISCH K. WEHH J. HERZ J. PREHX S. F&w R. VINGRON M. mechanisms that govern the assembly and disassembl) D~BHEUTEIN B: Homology of the 54K Protein of Signal- Recognition Particle, Docking Protein, and Two E. Coli of the translocon and the ribosome during the transloca- Proteins with Putative GTP-binding Domains. Nnflrre 1989, tion of pre-proteins across the ER membrane. 340:47u+82. 578 Membranes
11. BERNSTEIN HD, Porn MA, Smua K, HOEIEN PJ, BRENNER S. 2-I. Ron-m~&rr J& MEITR DI: Secretion in Yeast: Translocation WAGER P: Model for Signal Sequence Rkcognition from and Glycosylation of Prepro-a Factor in Vftro Can Occur Amino-acid Sequence of 54k Subunit of Signal Recogni- via ATP-dependent Post-translational Mechanism. ElfSO J tion Particle. Nufure 1989, 340:482486. 1986, 5:1031-1036.
12. ZOPF D, BERNSTEIN HD. JOHNSON AE, WALTER P: The 25. ROTHBUTT JA, Dfim~s RJ, S&?~IXRS SL, DAlIhi G, SCHEKMN Methionine-rich Domain of the 54 kD Protein Subunit of R: Multiple Genes are Required for Proper Insertion of Se- the Signal Recognition Particle Contains an RNA Binding cretory Proteins Into the Endoplasmic Reticulum in Yeast. Site and Can be Crosslinked to a Signal Sequence. ElfEO J Cell Rio1 1989. 72:61-68. / 1990, 9:4511-4517. 26. CHIRICO WJ. WATEK~ MG. BI.O~EL G: 70K Heat Shock Related 13. HIGH S, D~BBERVEIN B: The Signal Sequence Interacts with Proteins Stimulate Protein Translocation into Microsomes. the Methionine-rich Domain of the 54-kD Protein of Signal Xurltre 1988. 332:805-IO. Recognition Particle. J Cell Biol 1991, 113:229-33. 2’. DESHAIES RI. Ktx~ BD. WERNER WM. Clwc EA, SctiEKhwN R: A 14. ROMISCH K, WEBB J, LINGELBACH K. GAIISEPOHI. H. DOHDERSTEIN Subfamily of Stress Proteins Facilitates Translocation of Se- B: The 54-kD Protein of Signal Recognition Particle Con- cretory and Mitochondrial Precursor Polypeptides. Nuture tains a Methionine-rich RNA Binding Domain. J CeN Hi01 1988. 332:801X805. 1990, 111:1793-1802. 28. SCHlJ3SllniD~ G. ‘&~DhII’NDSSON G. Bow’hl.&V f1. ~IhlhWLZWNN R: A Large Secretory Protein Translocates Both Cotrans- 15. LCrrCKE H, HIGH S, ROhllsCH K, ASHFORD A. DOBHERVEIN B: IationalIy, Using Signal Recognition Particle and Ribosome. . . The Methionine-rich Domain of the 54 kDa Subunit of Signal Recognition Particle is Sufficient for the Interaction and Post-translationally. without These Ribonucleoparticles, When Synthesized in the Presence of Mammalian Micro- with Signal Sequences. ElfSO ./ 1992, 11:15i3-1551. By photo-crosslinking, free SRP5+ mzq shown to interact nit11 signal somes. .I Biol Chw 1990. 265:1396(r-13968. sequences. The M.domain alone also was shonn to be active in signal 29. elms DC. PORN% MA. YC’titn-:~ I’: Succburon~~yces cerevisiue sequence binding Alkylation of the C&domain of SRPSrt prments the and Schizosucchwonz~ces potnbe Contain a Homologue to binding of the signal sequence to the M.domain. which can be reversed the 54.kD Subunit of The Signal Recognition Particle That by proteolytic removal of the alkylated G-domain. This suaests that in S. cerevisiae Is Essential for Growth. ./ Ce// Hiol 1989. the G-domain is in contact nith the M-domain and may regulate the 109:3223-3230. interaction of the signal sequence with the M.domain. 30. A\IASA Y. N%%vo A. ITO K. MORI M: Isolation of a Yeast 16. ~AUFFER L, G-CIA PD, HARKINS RN, COUSSENS L. L~JRICH A. Gene, SRHl, that Encodes a Homologue of the 54K Subunit Wxtx~ P: Topology of the SRP Receptor in the Endoplas- of LMammalian SignaI Recognition Particle. .I Riochenr 1990. mic Reticulum Membrane. Au!zrre 1985, 318:33+338. 107:-157-+63.
17. BOLIRNE HR, SANDERS DA MCCOR?.!ICK F: The GTPase Super- 31. Ocr, S. Pofu-ri? M. \~‘AI.TER P: The Signal Recognition Particle family: a Conserved Switch for Diverse Ceil Functions. &cl- . . Receptor is Important for Growth and Protein Secretion lure 1990. 348:12+-132. in Succburomyces cererdsiue. .Ilol Rid Cdl 1992, in press. Deletion of the gene encoding the S. cerel,isiue homolog of the SR a- 18. GI~MORE R, BLOBEL G: Translocation of Secretory Proteins subunit resulted in \iahle. hut poorly groming cells. Depletion of the Across the Microsomal Membrane Occurs through an En- SR a-s&unit caused precursors of secretov and membrane proteins to vironment Accessible at Aqueous Perturbants. Cc,// 1985, accumulate in the c?n)sol: different pre-proteins are affected to different 42497-505. degrees.
19. CONNOUY T, GILMORE R: The Signal Recognition Particle Re- 32. lt\ss DC. W’AI:I%R P. The Signal Recognition Particle in S. ceptor Mediates the GTP-dependent Displacement of SRP ceretrisiue. Cell 1991, 67:131-l-13 from the Signal Sequence of the Nascent Polypeptide. Cell %&on of the .VU%gene of S. cerelvkiue resulted in \iahlr. hut poorl! 1989, 57:599-&O. groning cells. The depletion caused precursors of secretor? and mem. brdne proteins to accumulate in the c)%)sol, but different pre-proteins 20. CONNOUY T. RAPIEJKO PJ. GIUIORE R: Requirement of GTP are affected to ;I different degree. . . Hydrolysis for Dissociation of the Signal Recognition Par- ticle from Its Receptor. Science 1991. 252:1171-1173. 33. A\ti~a Y, Nwo A: SRHl Protein, the Yeast Homolog of A high affinity salt-resistant complex between SRP and SR forms in the . the 54 kDa Subunit of Signal Recognition Particle, is In- presence of the non-hydrolyzable analog of GTP, Gpp( NH )p, indicating volved in ER Translocation of Secretory Proteins. fTBS Len that GTP hydrolysis is required for the release of SRP from its receptor 199 1, 283:325-328. and recycling of these components in translocation. Depletion of SRP5-r homolog of S. cerecvkirce resulted in the accumu- lation of precursors of secretov proteins. 21. RA!-YEJKO PJ, GI~MORE R: Protein Translocation Across the . Endoplasmic Reticulum Requires a Functional GTP Bind- 3-1. STllUJNG CJ. Ronlmrr J, I lo5OI3l!clll M, Dmwe> R, SCHEKhlAN ing Site in the a-subunit of the Signal Recognition Particle . . R: Protein Translocation Mutants Defective in the Insertion Receptor. J Cell Biol 1992, 117:493503. of Integral Membrane Proteins into the Endoplasmic Retic- Mutations in the GTP,binding consensus sequences of the SR a-subunit ulum. Mel Biol Cell 1992, 3:12’+112. produced SRs that were impaired or inactimted in protein translocation Mutants that were defective in the insertion of membrane proteins were and their ability to form a Gpp(NH)p.dependent salt stable complex obtained by a selection that used a gene fusion of histidinol dehydro- with SRP, indicating that the GTPase activity of the SR a.subunit is re- genase and an integral membrane protein, hydroxylmethylglutary~ CoA quired for ttanslocation. reductase, as the targeting domain. Two temperature-sensitive lethal mutants in two complementation groups were isolated: a new allele 22. 0% S, NUNNARI J, MILLER J, P WALTER: The Role of GTP in of sec61 and a mutation in a nm gene sec65. . Targeting and Translocation. In Membrane Biogenesk wrd Protein Targeting. New Comprehensive Biochemistry. Edited 35. STIRIJNG CJ, HEUL’ITT EW: The Succhuromyces cereuisiue by Neupert W, till R. Amsterdam: Elsevier; 1992:123-136. . . SEC65 Gene Encodes a Component of the Yeast Signal Reviews the role of GTP in protein translocation and presents a spec- Recognition Particle with Homology to Human SRPl9. Nu- ulative model. lure 1992. 356:531-537. The DNA sequence of the SEC65 gene suggests that its protein is an 23. HANSEN W, GARCLA PD, WALTER P: In Vitro Protein Transloca- S. cere!Gue homolog of SRP19. Deletion of the SEC65 gene resulted tion Across the Yeast Endoplasmic Reticulum: ATP-depen- in liable hut poorly growing cells that exhibit translocation defects. An dent Post-translational Translocation of the Prepro-a Factor. extrdgeneic suppressor of sec65. I was cloned and is identified as the Cell 1986, 45397-406. SRp54 gene, consistent with the observations of Hann e/ NI. [36**]. Protein translocation apparatus in the endoplasmic reticulum Nunnari and Walter 579
36. HANN B, STIRUNG CJ, WALTER P: SEC65 Gene Product is a 47. HARTMANN E, WE~DMANN M. RAPOPORT TAz A Membrane Com- . . Subunit of the Yeast Signal Recognition Panicle Required ponent of the Endoplasmic Reticulum that May be Essential for Its Integrity. Nufure 1992, 356:532-533. for Protein Translocation. EMBO J 1989, 8:222+2229. The Sec65pgene product is shown to be a subunit of S. ceretWaeSRP. 43. CORUCH D. HARTLIANN E, PREHN S. RA~OPORT TA A Protein of A mutation in Sec65P disrupted the integrity of SRP, specifically caus- . . the Endoplasmic Reticulum Involved Early in Polypeptide ing SRP54 to dissociate from the particle. Overproduction of SRP54p Translocation. Nature 1992, in press. suppressed both the ts lethal and the translocation defect phenotypes Crosslinklng studies revealed a membrane glycoprotein in the vicinity of sec65. I mutant cells. of translocadng nascent chains. Using crosslinklng and reconstitution 37. HE F. BECKERICH J-M, GAILLUUXN G: A Mutant of 7SL in approaches, this protein, termed TRAM, was purified and shown fo be . Yurrowia hpolyticu Affecting the Synthesis of a Secreted stimulate? or required for the trdnslocation of several secretory pro- Protein. J Biol Ujem 1992, 267~1932-1937. teins. KELURIS KV, BowN S, GIUIORE R: ER Translocation Interrne- 38. YAVER DS, hk4TOBA S. ORGRYDZIAK DM: A Mutation in the 49. . Signal Recognition Particle 7S RNA of the Yeast Yarrowia . diates Are Adjacent to a Non-glycosylated 34-kD Integral Lypolytica Prefers Affects Synthesis of the Alkaline Extra- Membrane Protein. J Cell Biol 1991, 114:21-23. cellular Protease: in Viuo Evidence for Translational Arrest. Nascent secretory and membrane proteins in the process of transloca- J Cell Biol 1992. 116:60%616. tion and integration are shonn to crosslink to a 34 kD non-glycoprotein 137’1 and [38-l demonstnte that mutations in one of the two genes ER memhrme protein. encoding the 7SL SRP.RNA result in us lethal phenorypes and non.per- 50. THRIFT RN, ANDREW’S DW. WALTER P, JOHNSON AE: A Nascent missive temperdtures cause a specific decrease in the .synthesis of a se- . . Membrane Protein is Located Adjacent to ER Membrane creted prolein. These findings suggest that the mutant SW can function Protein throughout Its Integration and Translation, J Cell in translational arrest hut not transltx&on. Eiol 1991, 112:809-821. This stu* demonstrates that membrane proteins in the process of inte- 39. SlhlON SM, BLOW. G. ZIh~hmtlERG J: Large Aqueous Channels gration are photocrosslinked to swerdl newly identified ER membrane in Membrane Vesicles Derived from the Rough Endoplas- proteins until protein synthesis is terminated. These findings suggest mic Reticulum of Canine Pancreas or the Plasma Mem- that the rih)some is involved in compledng membrane protein integra- brane of Escherichia coli. Prcc Nctll Accid Sci LISA 1989. tion. 86:617&80. 51. ROBINSON A, WES~WOOD OMR, ALISTEN BM: Interactions of 40. SIMON SM, BLOBEL G: A Protein-conducting Channel in the . Signal Peptides with Signal Recognition Particle. Bicdwm J . . Endoplasmic Reticulum. Cell 1991, 65:371-380. 1990. 266:1+?156. Rough ER vesicles were fused to planar lipid hilayers and conductance Addition of s)nthetic signal sequence reversed SRP.dependent elonga- across the membrane ~3s measured. Vectorial discharge of trdnslocar- tion arrest and photwrosslinked 10 both SRI’54 and SRI%8 A 45kD ing nascent chains using puromycin led to a large increase in membrane ER membrdnr.associated protein is also observed fo crosslink to the conductance. Dissociation of the rihosome from the membrane with .synthedc signal sequence. the addition of salt abolished conductance. indicating that observed conductance was dependent on the interaction of the ribosome with 52. ~IHICH G, UIUCH B. SARATINI D: Proteins of Rough Mi- the memhrane. crosomal Membranes Related to Ribosome Binding 1. Identification of Ribophorins I and II, Membrane Pro- -11. NICCCI~I-~A CV. BLOBEI. G: Assembly of Translocation com- teins Characteristic of Rough Microsomes. J Cell Biol 1978, petent Proteoliposomes from Detergent Soiubilized Rough 77:&i--r87. Microsomes. Cell 1990, 60:25+269. 53. HOKTSCH M. A~OSSA D, ME~ZR D: Characterization of Se- 42. NICCHITI-A C. MKLLKCIO G, BIL)HEL G: Biochemical Fraction- cretory Protein Translocation: Ribosome-Membrane In- . ation and Assembly of the Membrane Components that teraction in Endoplasmic Reticulum. J Cell Biol 1986, Mediate Nascent Chain Targeting and Translocation. Cell 103:241-253. 1991, 65:587-598. 54. YOSHIDA H. TOND~KORO N. ASANO Y. MIZL~AWA K, YA~UG~SHI Total ER detergent extracts were fractionated; the resulting fractions R. HORIGO~!E T. S~IG;PNO H: Studies on Membrane Proteins had no translocation activity when assayed separately hut translt~ation Involved in Ribosome Binding on the Rough Endoplasmic restored when the fractions were combined Reticulum. Biochem J 1987. 245:811-819. 43. MICL~ACCIO G, NICCH~I-~A C. BLOBEI. G: The signal sequence 55. Yu Y. S~ATINI D, KREI~ICI~ G: Antiribophorin Antibod- . . receptor, unlike the signal recognition particle receptor, ies Inhibit the Targeting fo the ER Membrane of Ribo- is not essential for protein translocation. J Cell Biol 1992. somes Containing Secretory Polypeptides. J Cell Biol 1990, 117:1525. 111:1335-1342. Detergent extracts were immunodeplered of either SR or SSR and used in a reconstitution assay. Vesicles depleted of SR were unable 10 target. 56. KELKHER D. KRE~BICH G. Gilhroa~ R: Oligosaccharyltrans- translocate or integrate nascent secretory and membrane proteins. In . . ferase Activity is Associated with a Protein Complex contrast, these actitiries were untiected in vesicles derived from SSR- Composed of Ribophorins I and II and a 48kD Protein. depleted extracts. These findings indicate that SSR does not function in CeN 1992, 69:1-11. a rate.limiting step in protein translocation. Oligosaccha~ltransferase activiv from canine pancreas ER co-purifies wivith a protein complex composed of 66, 63 and 18 kD subunits. The 44. WlEDhIANN M. KKUXLIA TV, HARTuNN E, R\POI’ORT T.4: A 66 and 63kD subunits are identical to ribophorin I and II. which are Signal Sequence Receptor in the Endoplasmic Reticulum found in the L-icinity of the translocon. Ribophorin 1 contains a dolichol Membrane. Nrrrrrre 1987, 328:830-832. recognition consensus sequence.
45. KRIEG UC, JOHNSON AE, WALTER P: Protein Translocation 57. SA\ITZ AJ. MEYER DI: Identification of a Ribosome Recep- across the Endoplasmic Reticulum Membrane: ldentilica- tor in the Rough Endoplasmic Reticulum. Nnfure 1990, tion by Photo-crosslinking of a 39-kD Integral Membrane 346:54&544. Glycoprotein as Part of a Putative Translocation Tunnel. J TAZAU’A S. UNL:hbi M. TOND~KORO J, A%NO Y, OHSUMI Cell Biol 1989. 109:203%20-13. 58. . T. ICI-II,WIHA T. SL~GANO H: Identification of a Membrane 46. G~~UICH D. PREHN s, HARThIANN E. HER% J, OTTO A, m-r Protein Responsible for Ribosome Binding in Rough Mi- R, WEIDh(ANN M, &-4EsPEL S, DOBHER 59. NLINNARJ J, 1.. ZD, OCG SC, WALTER P: Chqcterization of 67. ZI~IXII~RXIAN DL, WAII~IX I’: Reconstitution of Protein Translo- . Rough Endoplasmic Reticulum Ribosome Binding Activity. cation Activity from Partially SolubiIized MicrosomaI Vesi- Nutrrrc 1991, 352:63-o. cles. J Hiol 0ettr 1990. 265:&&-1053. Ribosome-binding activiv quantitatively soluhilized does nor co-frac- hlrlscii A+ WIE~MANN M. RAWI~OH’I’ T: Yeast SW Proteins In- tionate with the 180kD protein proposed 10 Ix the ribosome rc- 68. ceptor [57]. In contrast to the 180 kD, the rihosome.hinding acritic . . teract with Polypeptidcs Traversing the Endoplasmic Retic- co.fractionates with a significandy smaller, positivcty charged prorein. ulum Membrane. Cell 1992, 69:3+352. These ohsenqtions indicate that the 180 kD protein is not rcsponsihle I~hc~tocrosslinkin~ of translocation intermediates, formed using ribo- for ribosome-binding activiy. This conclusion is supportcxl by the lind- some-associarrd truncated nascent chains. indicate that %x61 p is in the ings of Collins and Gilmore [ 60.1. xicinit), of the translocating n:Lscenr chain. W/hen shorter nascent chain intermediates are emI~loycd. crosslinking to Sec62p is also ohsen&. 60. COIUNS P. CILMXE R: Ribosome Binding to the Endoplasmic Crosslinks 11) Scv62p are abolished by acldirion of ATP. indicating that . Reticulum: a 180.kD Protein Identified by Crosslinking to ~echlp acts hefore Scc6I p. Membrane-bound Ribosomcs is not Required for Rihosome Binding Activity. J Cdl Rid 1991, 114:6396+9. 69. S~IXK\ 5. WI~II’~-II~IJ~ K. \‘OGEI. J. Rose M, S(:HEtihl,W R: Sec61p Ribosomes in the process of rr.1nskxxtion crosslink to sever,ll ER men1 . . and BiP Directly Facilitate Polypeptidc Translocation into hrxne proteins, including SR. rihophorin I and SSHrr. The l~rrck~min:1nt the ER. Cell 1992. 69:353-365. crosslink product was a 180 kD prorcin that \va.s proposed to lx ;I I-- SC~~I I> crc~sslinkcul in an ATP-depenclcnt manner 11) ;I translocation in hosome receptor [ 571. Rihosome-binding activiy, however. could he ~crmedia1e formed using a l~r~l~r~~-a-t:.1ctt,r-a\iclin chimeric protein. Mu fmctiWXWd from rhe 180 kD protein. indicating thar the 180 kD pro- rxions in SecO?p and .‘+~O.~/J inhibir crosslinking 10 .Sc,cb /p, whrrcah tein does not function as a rihosomc receptor. Thih conclusion 1s in mur;ltions in A?w,$ do n111 alfccr crc&inking of the uxnsk)ca~ion ill- agrcwment tith the suwestions of Nunnari r/ N/. [ 59-I. termciliate to .SecC,fp to such an cstent. suggesting that rhe Str62p and Sec63p function before. and KarLI> protein hlnctions after. Secbl I> in 61. %IhlhlEffiu~~ D. W.Wi:H I’. An ATP-binding Membrdnc Protein Irxlblocalion. . Is Required for Protein Translocation Across the Endoplas- mic Reticulum Membrane. C@// k’e 66. Yl1 Y, ZHANC Y. SAHATINI DD, KRI%KH G: Reconstitu- tion of Translocation-competent Membrane Vesicles from Detergent-solubihzed Dog Pancreas Rough Microsomes. J Nunnan. I’ Waher. Depanmenr of Hiocl~emisrry and Riophysics. 1 Ini Proc Null Acud Sci (IS.4 1989. 869931-9935. verhiv of California. San Fr.cncisc~~. California 9~1+3-0+#. LISA.