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Summary The synthesis of biological membranesrequires the insertion of proteins into a lipid bilayer. The rough endoplasmic reticulum of eukaryotic cells is a principal site of membrane biogenesis. The insertion of proteins into the membrane of the endoplasmic reticulum is mediated by a resident proteinaceous machinery. Over the last five years several different experimental approaches have provided information about the components of the machinery and how it may function. Introduction The eukaryotic cell is made up of a number of highly specialised, membrane enclosed, compartments. How proteins are targeted to their correct destination within the cell and how these proteins are then inserted into or completely translocated across the impermeable membrane barrier of the target compartment are questions which are central to our understanding of cell biology. It is now clear that the protein itself contains the signal which directs it to a specific compartment within the cell (Fig. 1). The first such signal to be defined was the endoplasmic reticulum (ER) targeting signal('). Subsequently other signals rcsponsible for targeting to mitochondria. peroxisomes and the nucleus were defined(*s3).
Newly synfhesked proteins
Secretory pathway
* CNucleus)
I\(-] Mitochondrion)
.
Fig. 1. Possible subcellular destinations for newly synthesised proteins preqent in the cytosol of eukaryotic cells.
NH2 terminus of the nascent chain and their function is to target the protein to the EK and then initiate its translocation across the membrane. During the translocation process these signals are cleaved from the nascent chain by the signal peptidase complex(6)present at thc lumenal side of the ER membrane. In thc absence of any further signals the nascent chain will be completely translocated across the membrane into the lumen of the ER. Membrane proteins with a clea~ablcsignal sequence also possess a 'stop transfer' sequence which terminates the translocation of the nascent chain before i t is complete and acts as the transinenibrane anchor for the memSignal-anchor sequences
n
Cleavable signal sequences
N C Signal Sequences cylosol The essential feature of the signal sequences which direct Membrme I I, 1 c v SPzse proteins to the ER is a linear stretch of 7 to 20 apolar amino < N I acid residues present within the nascent polypeptide Secreted L hai in(^,^). Such signals are present on a large number of membrane and secretory proteins, the ER being the major entry point into the secretory pathway (Fig. 1j. The majority of membrane proteins resident in both organelles throughout Fig. 2. The types of ER targeting signal sequences present within the secretory pathway, and the plasma membrane, are membrane proteins and their subsequent orientation in thc ER inserted into the lipid bilayer at the ER. Secreted proteins, membrane. Filled regions represent signal-anchor (SA) or clcavwhich are ultimately destincd to exit the cell completely, are able signal sequences as indicated. ST indicates a stop-transfer also translocated across the ER membrane and enter the sequence. Arrows indicate signal peptidase (SPase) cleavage sites. lumen (Fig. 2). * indicates an acceptor site for Asn linked oligosaccharide and the The ER targeting signals are of two types: cleavable signal branched side chain shows that addition o f the high mannose carbosequences and uncleaved signal-anchor s e q ~ e n c e s ( ~ . ~ ) ( s e ehydrate moiety has occured. Type I proteins have their NH2-termini Fig. 2). Clcavable signal sequences are found on both memextracytoplasmic while type I1 proteins have their NH2-termini brane proteins and secreted proteins. They are present at the cytoplasmicallylocated. L"11,W
d
r
/
brane protein. Stop transfer sequences usually consist of a linear stretch of some twenty apolar amino acid residues followed by several positively charged residues. Signal-anchor sequences are present within membrane proteins and serve to both target the protein to the ER and then to anchor the protein stably into the lipid bilayer. A signal-anchor sequence can be present anywhere within the nascent chain of a membrane protein.
Targeting The cell identifies ER targehng signals as such by virtue of a cytosolic adaptor - the signal recognition particle (SRP)(7.8). SRP recognises and binds to the ER targeting signal a5 it emerges from the ribosome as part of the growing nascent chain. The resulting nascent chain/ribosome/SRP complex will then exclusively bind to the ER membrane where the docking protein (or SRP receptor), a specific receptor for the complex, is located (Fig. 3). Signal sequence recognition occurs via an interaction between the methionine rich, COOH-terminal, domain of the 54 kDa subunit of SRP (SRP54) and the hydrophobic signal s e q ~ e n c e ( ~Having j. bound to a signal sequence the role of the SRP is two fold: 1) to maintain the nascent chain in a translocation competent ( i t . unfolded) state compatible with membrane insertion(lO,ll);2) to tag the nascent chain as destined for the ER. Upon arrival of the nascent chain/ribosome/SRP complex at the ER the signal sequence is released from SRP54 in a mechanism which requires both the docking protein complex (a and p subunits) and GTP(12,13).While the release of the nascent chain does not require GTP hydrolysis the subsequent release of the SRP from the docking protein (Fig. 3 ) .
-
N
signal-anchor sequence SRP: signal ncogniiion panicle DF: docking prolein TI & T2: components of trmslmlion machinery.
Fig. 3. SRP mediated ER targeting cycle. SRP binds to the signal sequence of the growing nascent chain and targets the nascent chainlrihosomelSRP complex, to the ER membrane. Upon interaction with the docking protein complex, the nascent chain is released from SRP and interacts with the translocation machinery. SRP is released from DP upon GTP hydrolysis and rejoins the cytosolic pool. The synthesis of the nascent chain is completed at the membrane and the protein is stably inserted into the lipid bilayer.
Membrane insertion After the release of the nascent chain from SRP, it interacts with the ER translocation machinery. The nascent chain now has two possible fates (Fig. 2): 1) it can be completely translocated across the ER membrane and enter the lumen as a secretory protein('.l5); 2) a selective translocation of only part of the protein can occur leaving the other region cytosolically exposed and the protein stably inserted in the membrane. Thus during membrane insertion a partial rather than a complete translocation of the nascent chain occur^(^^.^^)). The translocation event itself i s probably identical in both cases and the type of signal sequence present will determine the fate of the protein after translocation has been initiatedcs,17) (Fig. 2). The final orientation which a membrane protein assumes in the lipid bilayer is dependent upon the kind of signal sequence it p o s s e s ~ e s ( ~Proteins ~ ~ ) . which span the membrane once and have a cleavable signal sequence always adopt a type I orientation, the NH2 terminus being extracytoplasmic (Fig. 2). Signal-anchor proteins which span the membrane once can adopt either a type I or a type I1 oricntation (Fig. 2). The orientation of signal-anchor proteins is determined b y the properties of the nascent chain and in particular the number of charged residues present in the two hydrophilic regions adjacent to the hydrophobic core of the signal-anchor s e q u e n ~ e ( ~ ~On ~ ~ ' the ~ ) . basis of theoretical consideration~('9,*~), and supporting experimental a mechanism where thc nascent chain inserts into the membrane in a loop conformation (e.g. Fig. 3) is currently f a v o ~ r e d17). (~~~~
The Generation of Translocation Intermediates The environment of the nascent chain during membrane insertion has been investigated by several approaches. A prerequisite of these investigations has been the ability to trap the nascent chain during the process of membrane insertion and generate 'translocation intermediates'. The underlying assumption is that the interactions of these translocation intermediates with components of the ER are the same as those made by the nascent chain during the normal course of membrane insertion. Such translocation intermediates can be made by using an artificially truncated mRNA to prime the in vitro synthesis of the nascent chain under study(22).Since the tmncated inRNAs lack a stop codon the ribosome stalls at the 3' end of the mRNA and thc resulting incomplete nascent chain remains stably associated with the ribosome. If translation is carried out in the presence of SRP and rough microsomes (ER vesicles) the protein is correctly targeted to and inserted into the microsomal membranc. Howcver, since thc COOH-terminus of the nascent chain remains attached to the ribosome, the nascent chain is jammed in the translocation site(23)(Fig. 4).
Cross-Iinking A direct method to analyse the immediate environment of these translocation intermediates has proved to be cross-link-
Table 1. Summary of ER proteins which are crosslinked to ing. Since the nascent chains can be made in vitro they can trunslocation intermediules easily be radioactively labelled. If the labelled nascent chain is then covalently cross-linked to a second, unlabelled, procrosslinked to proteins with: tein (Fig. 4), the resulting cross-linking product can be detected by the appearance of a radioactive product which is cleavable Signal-anchor M.wt (ma) signal sequence sequence Reference larger than the original nascent chain. Since the nascent chain is trapped during the act of translocation it is reasonable to Non-glycoproteins believe that proteins which can be cross-linked to these 125I imp34 34 yes nd P37 37 Yes yes [26]& I translocation intermediates are candidates for components of the translocation machinery. GIycoproteins SSRa 35 Yes yes [26,41] Two methods have been used to cross-link translocation TRAM 36 ycs nd I1 intermediates to adjacent ER proteins. In the first ‘phomp39 39 Yes yes [23,27] tocross-linking’ approach, a modified lysine residue is incorporated into the nascent chain during in vitro synthesis. Upon nd=not determined ultraviolet irradiation the side chain of the modified lysine I=S. High unpublished data II=T.A. Kapoport pers. comm. forms a highly reactive radical which will rapidly react with NB. mp39 is equivalent to SSRa and TRAM. imp34 and P37 are nearby proteins(24). A second approach has been to use probably the same coinponent. bifunctional cross-linking reagents, which react specifically only with certain amino acid side chains, and add these to the translocation intermediates generated as d e ~ c r i b e d ( ~ In ~.~~). tors. These include the nascent chain used, the length of the both cases only a very small number of cross-linked products nascent chain in the translocation intermediate and the crossare observed consistent with only a small subpopulation of linking method. We have recently found that the exact posiER proteins being in the immediate vicinity of the nascent tion within the nascent chain from which cross-linking chain translocation intermediate. occurs also influences the ER protein which is crosr-linked The specificity of this interaction between the nascent (S. High, B. Martoglio, J. Brunner and B. Dobberstein, chain and ER proteins has been tested by determining the unpublished data). Thus, different regions of the nascent cross-linking partners of the nascent chain after the translochain of a translocation intermediate are in contact with difcation intermediate has been disrupted by removal of the ferent ER proteins. To date cross-linking has identified 34 ribosome. This can be accomplished by treatment of the riboand 37 lcDa non-glycoproteins and at least two 35-36 kDa some with EDTA or puromycid2@,or by allowing the synglycoproteins as candidates for components of the translocathesis of the complete protein rather than a truncated vertion machinery (see Table I). So far only a 36 kDa glycoproion(^^). In both cases, although the nascent chain clearly tein denoted ‘TRAM’ has been shown to play a direct role in remains within the membrane, it is no longer found crosstranslocation (sce below). linked to the proteins observed when the translocation intermediates are used. Thus, the proximity of the nascent chain to these ER components is transient and dependent upon a Indirect Analysis ribosome-associated translocation intermediate. The biochemical properties of translocation intermediates The major proteins which have been identified by crosshave also been examined in an attempt to determine their linking approacheb are shown in Table 1. The pattern of ER environment. Translocation intermediates of the secreted proteins which is cross-linked is dependent upon several facprotein preprolactin have been found to be extracted from the membrane by agents such as urea. This suggests that at this stage the nascent chain may not be completely integrated into the lipid bilayer but rather in an aqueous environment or in contact with other proteins(28).When translocation intermediates are solubilised at high detergent concentrations, the nascent chain is significantly protected from added protease, including NHz-terminal regions exposed outside the riboCytosol some. This suggests that after solubilisation of the membrane the protein components of the translocation site stay associated with the membrane inserted region of the nascent chain Membrane and protect it from the added protease(2Y).
Lumen Fig. 4. A ribosome bound translocation intermediate.
* indicates a
residue of the nascent chain suitable for cross-linking to adjacent components. The hatched column represents an ER protein in proximity to the translocation intermediate.
Insertion into the ER Requires Protein Components A direct role for ER proteins in mediating membrane insertion and translocation is indicated by the observation that some covalent modifications of ER component{ can com-
pletcly block these processes. It has been shown that an NEM sensitive protein component is required for correct translocation in ~ i t r d ~This ~ ) component . is clearly distinct from the docking protein which is also sensitive to NEM and is necessary for correct targeting (see above) - a prerequisite for membrane insertion. Recently it has been found that the covalent modification of ER membrane components with the ATP analogue 8-azido ATP also blocks both the translocation of secreted p r o t e i n ~ ( ~and ' , ~ the ~ J insertion of membrane proteins (S. High, unpublished data). It is not presently known which components of the translocation machinery are sensitive to these modifications.
Genetic Analysis A genetic \election has been used with the yeast Sacchnmmyces cerivisiae to allow the identification of genes which encode proteins that are required for the translocation of proteins across the membrane of the ER(33.34).Three integral membrane proteins of the ER were identified: Sec6lp, Sec62p and Sec63p. When these proteins are absent or defective the efficiency of translocation of secretory proteins acros~the ER is much r ~ d u c e d ( ~ ? -Sec6lp, '~) Sec62p and Sec63p are found as part of a complex of proteins present in the Saccharomyces cerevisiae ER(36Jand the evidence that this complex forms at least a part of the membrane insertion machinery is compelling. While the suspicion is strong(?'), there is as yet no direct evidence that proteins homologous to Secblp, Sec62p and Sec63p are present in the mammalian ER.
is not essential for membrane insertion or tr'anslocation in vitrdS2).When antibodies against the other ER components identified by cross-linking are available, such reconstitution techniques will provide a useful method to test their role in membrane insertion and translocation.
Accessory Proteins In addition to proteins which are directly involved in membrane insertion and translocation, other proteins appear to be close to the site of translocation but not directly involved in the process per se. Antibodies directed against ribophorin I and SSRa have been shown to block the translocation activity of membrane^(^^,^^). Ribophorins I and I1 form a part of the complex responsible for the addition of asparaginelinked carbohydrate to nascent chains emerging on the lumenal side of the ER(44).SSRa has recently been shown to be a major calcium binding protein of the ER and its function is at present unclear.
The Machinery for Membrane Insertion There is now a wealth of data to show that ER proteins are required to mediate the insertion of nascent chains into the lipid bilayer. Current evidence also suggests that both membrane insertion and translocation use the same ER machinery. A number of possible types of translocation site across the lipid bilayer of the ER have been proposed: 1) A protein and lipid mediated translocation site(5).The hydrophilic regions of the protein which are translocated across the membrane would contact ER proteins while the hydrophobic signal sequence would interact directly with lipids. Reconstitution 2) A transient protein-lined translocation ~ i t e ( ~ , ' ~The ,l~). formation of the translocation site would be coupled to the In mammalian cells biochemical techniques have provided a arrival of a correctly targcted nascent chain complex at the way in which the role of an ER protein in membrane insertion ER. The translocated region of the protein would pass and translocation can be tested directly. It has been shown through the channel while the hydrophobic signal would be that the ER vesicles (rough microsomes) which are used to retained. study membrane insertion in vitro can be taken apart and subsequently put back together or r e c o n ~ t i t u t e d ( ~ ~The - ~ ~ ) . 3) A gated protein-conducting channel(46).The opening of the chaiiiiel would be closely regulated to ensure that only the reconstituted vesicles are capable of correctly translocating translocated portion of a nascent chain can normally pass secreted protcins into their l ~ m e n ( ~ * and - ~ ~of ) , correctly inserting membrane proteins into their lipid b i l a ~ e r ( ~The ~ ) . through. During the course of membrane protein insertion it is clear requiremcnt for any ER component in the translocation that the hydrophobic signal-anchor or stop transfer sequence process can thus be tested by removing it from the solubilised must exit the translocation site in the lateral plane and enter membrane fraction prior to reforming the vesicles. the lipid bilayer at some stage. Cleavable signal sequences If a lectin column is used to deplete the solubilised ER of may also exit the translocation site prior to cleavage by signal all glycoprotcins the resulting reconstituted membrane vesipeptidase although this is at present not known. The exit of a cles show a much reduced translocation activity("). It has hydrophobic signal raises two questions: how and when? recently been shown that the addition of a single purified glyHow the interaction occurs is straightforward to explain for a coprotein to the depleted mixture can restore the ability of the protein/lipid translocation site since lipid is already in close reconstituted vesicles to translocate certain secreted proteins. contact with the signal. In the case of a transient protein-lined The 36 kDa glycoprotein responsible for restoring the in site, its very nature would allow for a subunit to be displaced vitro translocation activity to the depleted vesicles has been at some point giving the nascent chain lateral access to the named the translocating chain-associating membrane prolipid bilayer('@. A protein-conducting channel would also tein(TRAM) (T.A. Rapoport, pers c o r n ) . Reconstitution have to allow lateral access to the lipid bilayer in order to has also been used to show that a previously identified 35 allow the stable integration of membrane proteins'46). kDa glycoprotein, named the signal sequence receptor If membrane insertion does occur initially in an all protein (SSRCX)(~]), although in proximity to some nascent chains environment, be it of a transient or a permanent nature, then during their membrane insertion and tran~location(~~,~~,~~,~l),
how is the message that the hydrophobic signal sequence should now enter the lipid bilayer conveyed? One possibility is that this is linked to the departure of the ribosome from the ER membrane upon the completion of protein synthesis. A similar mechanism has been proposed to act as a signal for closing a gated protein-conducting channel upon the completion of translocation("). Coupling the lateral exit of the hydrophobic sequence of a membrane protein from the translocation site to the departure of the ribosome is also consistent with cross-linking data. Thus, in the absence of the ribosome the previously identified contacts betwecn nascent membrane proteins and ER proteins are no longer o b ~ e r v e d ( ~ ~The 3 ~release ~). of the ribosome would allow a transient translocation machinery to dissociate, enabling the signal-anchor or stop-transfer sequence of a membrane protein to enter the lipid b i l a ~ e r ( ~., l ~ ) The evidence for the involvement of ER proteins in membrane insertion has already been discussed. None of these data, however, approach the question of what the physical make up of the translocation site looks like. One approach to this problem has been to use electrophysiological techniques to analyse the channels present in the ER. Using this approach, data consistent with the translocation site being a gated channel have been obtained'46).
The Membrane Insertion Site - a Model The work I have described leads me to make the following general conclusions about the machinery responsible for membrane insertion into the ER (see Fig. 5): 1) it is a complex formed from a number of heterologous protein subunits; 2) 'core' proteins of the complex are directly involved in the
process of membrane insertion and are absolutely required for function; 3) a number of accessory proteins are closely associated with the complex but do not play a direct role in membrane insertion. Rathcr, they are involved in related functions which normally occur during the membrane insertion process. Membrane insertion or translocation is only one step in a complicated journey which the protein makes. This includes targeting to the ER, insertion into the membrane or complete translocation across it into the lumen, and then subsequent sorting to the correct subcellular destination (see Fig. 1). The targeting machinery must be either closely associated with the translocation site or a part of it. Thus, after the release of the nascent chain from SRP54, it is directly transferred to ER membrane proteins(13).After insertion, the nascent chain emerges on the luminal side of the membrane. lf a suitable cleavage site is present, the NH2-terminal signal will be cleaved from the nascent ~ h a i n ( l $ ~ Both . ~ > ~secreted ). and membrane proteins are quickly modified by the addition of carbohydrate to suitable asparagine residuescs7). In addition the formation of disulphide bonds occurs rapidly implying that the catalytic activity of protein disulphide isomerase is close at hand(48'. Chaperones such as BiP may bind to the emerging nascent chain on the luminal side of the ER and mediate the correct folding, and assembly (in the case of oligomcric proteins), of the luminal portion of nascent membrane proteins(49).In yeast, there is evidence to suggest that BiP may also be directly required for efficient translocation to occur(S0,").
Conclusions The insertion of proteins into the membranc of the ER is mediated by a proteinaceous machinery. This translocation machinery consists of a protcin complex made up of scvcral heterologous subunits and is responsible both for the insertion of membrane proteins into the lipid bilayer and the complcte translocation of sccretory proteins into the lumen of the ER. While the specific nature of the proteins which make up the translocation machinery of mammalian cells remains unclear, several good candidates have now been identified. A detailed structure of the translocation complex and the mechanism by which translocation occurs would secm to be within reach.
I
pD1
J
Fig. 5. Model translocation site. The membrane bound ribosome is synthesizing a type I1 signal-anchor membrane protein. The hydrophobic core of the signal-anchor sequence is shaded black. DP = docking protein. T1 and T2 = protein components of the translocation machinery. SPasc = signal peptidase. CHO = oligosaccharyltransferase complex, of which ribophorins I and 11 form a part(44)). RR = putative ribosome SSR = signal sequence receptor (asubunit). BiP = heavy chain binding protein. PDI = protein disulphide isomerase.
Acknowledgements Many thanks to Reid Gilmore and Tom Rapoport for communicating results prior to publication. Thanks also to Sgren Andersen, Bernhard Dobberstein, Jonathan Howard, Joen Luirink, Henrich Lutcke and Jean Pieters for their careful reading of the manuscript and useful suggestions for its improvement.
Note added in proof The work on thc TRAM protein is now published, see Gorlich, D., Hartmann, E., Prehn, S. and Rapoport, T. A. (1992). Nature 357,47-52.
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29 Connolly, T.,Collins, P. and Gilmore, R (1989). Xccers of yrolrinrse K to parrially translociited nasccnt polypeptides in intact and dctergent-solubilized mcmbrancs. J. Cell Bid. 108. 299-307. 30 Ncchitta, C.V. and Blnhel, G. (1989). Nascent secretory chain binding and translocation aredistinct pruceascs: differentiationby chemical alkylation. 1. Crll Bin!. IO~,~SY-~Y~. 31 ~appa,P.,h.layinger,P., Pipkorn,IL,Zimmermann,Jf.andZimmermann,R. (1991). A microsomal protein is involved in ATPdependent transpm uT preswretory proteins into mammalian microsomes. EMB0.J. 10,2795-2803. 32 Zlmmerman, D.L. and Walter,P. (1991). An ATP-binding protein is required for pmteiii translocation across the endoplasmicreticulum. CeIlRegul. 2,851-859. 33 Deshaies, R. and S c B e h n , R (1987). A yeast mutant defective at an w l y stage in import uf secretory protein precursors into the endoplasmic reticulum. J. Cell B i d . 105.633-645. 34 Rothblatt, J.A., Deshaies, RJ., Sanders, S.I.., Darun, C. and Schekman, W. (1989). hfiiltiple genes are required for proper insertion of swretory protcins into the endoplarniic reticulum in yeast. J. Cell Biol. 109,2641-2652. 35 Deshaies. W .and Schekman, R. (1989). SEC62 encodcs a putative niemhrane protein q u i d for yrotcin translocation intn the yeast endnplasinic reticiilum. 1. Crll Bid. 109,2653-2664. 36 Deshaies, RJ., Sanders, S.L., Feldheim, D.A. and Schekmnn, R. (1991). Assembly ul' yeast SEC proteins involved in translocation into thc cndoplasmic reticulum into a mernbnne-bound multisubunitcnmplex. Xafurr 349.8oC1-8O8. . 37 Gilmnre, R. (1991). The protein translocationapparatus of the ruuph endoplasmic reticulum. its acwcialed pmteins, and the mechanism uf tran~lucatiun.Curr. Opin Cell ninl. 3,580-584. 38 Yu, Y., Zhang, Y., Sabatini, D.D. and Kreibich, C. (1989). Rcconstitution of Innslocation-competent incmbranc vesiclcs from dctergent solubilizcd dog pancreas roughmicrosomes. Proc. NaflAcud. Sci. USA 86.9931-9935. 39 Nicchitta, C.V. and Blobel, G. (1990). Assenihly of translncation-cumptent protenlipnsomesfrom dc.tergent-solubili7rdrough microsomes. Cell 60.259-269. 40 Zimmerman, D.L. and Walter, P.( I 990). Reconstitution or protein translocation activity from partially soluhiljmd micro~omalmembrane ve\icles. J. Biof. Clwm 265, 40.18-4053. 41 Wicdmann, hl., Kunchalia, T.V, lhrlmann, E. and Rapoport, T. (1987). A signal sequence receptor in lhe endoplasmic reticulum membrane. h'uture 328,830833. 42 Yu, Y., Sabatini, D.D. and Kreibich, C. (1990). Antiribophorin antihodies inhihit the largeling to the ER membrane of rihiwmes containing nascent stmtury polypeptides.J. CellBiol. 111, 1335-1342. 43 Ilartmann, L, Wicdmann, M. and Rnpoport, T.A. (1989). A membrane componcnt of the endoplasmic reticulum that may be essential for protcin translocation. EMBO. .I. 8.2225-2229. 44 Kelleher, D., Kmibich, G. and Gilmore, R. (1992). Oligosaccharyltrunsfera~e is iissociated with a protein complex composed of ribophorins I and I1 and a 48 k D protcin. Cell. 69.55-65. 45 Wad& I,Rindress,D., Caniernn,P.H., Ou,W-J., Doherty 11,JJ., Lnuvard,D., Rell, A.W., Dignard, D., Thomas, D.Y. and Reperon, J.J.M. (1991). SSKa and assuciated cdnexin are m j u r calcium binding proteins of the anduplamic reticulum membrane. J. Bid. Chem 266.19599-19610. 46 Simon, SM. and Blobel, C. (1991). X protcin-conducting chiinncl in the endoplarmic reticulum. Crll65.371-380. 47 Kornfeld, R. and Kornleld, S. (1985). Assembly of asparagine-linked uliguwcchiiridcs.Annu. Rev, Riochrm 54.63 1-664. 18 Freedman, R.B. (1989). Protein disulphide isomerase: multiple roles in the modification of nascent secretory proteins. Cel157,1069-1072. 49 Gething, hf.-J. and J. Sanibrook (1992). Protein folding in the cell. Nuture 355, 33-45. 50 Vogel, J.P., hlisra, LBI. and Rose, hLD. (1990). Loss of BiWGRP78 function hlocks tranhcatinn of secretoryproteins in yeacr ./. Cell B i d . 110. 1885-1895. 51 hieyer, 0.1. ( I 991). Protein trmslocatinn into the endopl~sniicreticulum: a light at theend ufthe timnel. Trends in CrlllhX 1,154-159. S2 hligliaccio, C., Niccbith, C.V. and Blubc~I,C. (1992). ' I l e signal sequence receptor. unliie the signal recognition panicle receptor. is not essential fur protein translocation.J. Cell Biol. 117, 15-25.
Stephen High is at thc European Molccular Biology Laboratory. Meycrhofstrasse 1. Postfach 102209,6900 Hcidclbcrg, Germany.
t t
(complex]
t
Summary The synthesis of biological membranesrequires the insertion of proteins into a lipid bilayer. The rough endoplasmic reticulum of eukaryotic cells is a principal site of membrane biogenesis. The insertion of proteins into the membrane of the endoplasmic reticulum is mediated by a resident proteinaceous machinery. Over the last five years several different experimental approaches have provided information about the components of the machinery and how it may function. Introduction The eukaryotic cell is made up of a number of highly specialised, membrane enclosed, compartments. How proteins are targeted to their correct destination within the cell and how these proteins are then inserted into or completely translocated across the impermeable membrane barrier of the target compartment are questions which are central to our understanding of cell biology. It is now clear that the protein itself contains the signal which directs it to a specific compartment within the cell (Fig. 1). The first such signal to be defined was the endoplasmic reticulum (ER) targeting signal('). Subsequently other signals rcsponsible for targeting to mitochondria. peroxisomes and the nucleus were defined(*s3).
Newly synfhesked proteins
Secretory pathway
* CNucleus)
I\(-] Mitochondrion)
.
Fig. 1. Possible subcellular destinations for newly synthesised proteins preqent in the cytosol of eukaryotic cells.
NH2 terminus of the nascent chain and their function is to target the protein to the EK and then initiate its translocation across the membrane. During the translocation process these signals are cleaved from the nascent chain by the signal peptidase complex(6)present at thc lumenal side of the ER membrane. In thc absence of any further signals the nascent chain will be completely translocated across the membrane into the lumen of the ER. Membrane proteins with a clea~ablcsignal sequence also possess a 'stop transfer' sequence which terminates the translocation of the nascent chain before i t is complete and acts as the transinenibrane anchor for the memSignal-anchor sequences
n
Cleavable signal sequences
N C Signal Sequences cylosol The essential feature of the signal sequences which direct Membrme I I, 1 c v SPzse proteins to the ER is a linear stretch of 7 to 20 apolar amino < N I acid residues present within the nascent polypeptide Secreted L hai in(^,^). Such signals are present on a large number of membrane and secretory proteins, the ER being the major entry point into the secretory pathway (Fig. 1j. The majority of membrane proteins resident in both organelles throughout Fig. 2. The types of ER targeting signal sequences present within the secretory pathway, and the plasma membrane, are membrane proteins and their subsequent orientation in thc ER inserted into the lipid bilayer at the ER. Secreted proteins, membrane. Filled regions represent signal-anchor (SA) or clcavwhich are ultimately destincd to exit the cell completely, are able signal sequences as indicated. ST indicates a stop-transfer also translocated across the ER membrane and enter the sequence. Arrows indicate signal peptidase (SPase) cleavage sites. lumen (Fig. 2). * indicates an acceptor site for Asn linked oligosaccharide and the The ER targeting signals are of two types: cleavable signal branched side chain shows that addition o f the high mannose carbosequences and uncleaved signal-anchor s e q ~ e n c e s ( ~ . ~ ) ( s e ehydrate moiety has occured. Type I proteins have their NH2-termini Fig. 2). Clcavable signal sequences are found on both memextracytoplasmic while type I1 proteins have their NH2-termini brane proteins and secreted proteins. They are present at the cytoplasmicallylocated. L"11,W
d
r
/
brane protein. Stop transfer sequences usually consist of a linear stretch of some twenty apolar amino acid residues followed by several positively charged residues. Signal-anchor sequences are present within membrane proteins and serve to both target the protein to the ER and then to anchor the protein stably into the lipid bilayer. A signal-anchor sequence can be present anywhere within the nascent chain of a membrane protein.
Targeting The cell identifies ER targehng signals as such by virtue of a cytosolic adaptor - the signal recognition particle (SRP)(7.8). SRP recognises and binds to the ER targeting signal a5 it emerges from the ribosome as part of the growing nascent chain. The resulting nascent chain/ribosome/SRP complex will then exclusively bind to the ER membrane where the docking protein (or SRP receptor), a specific receptor for the complex, is located (Fig. 3). Signal sequence recognition occurs via an interaction between the methionine rich, COOH-terminal, domain of the 54 kDa subunit of SRP (SRP54) and the hydrophobic signal s e q ~ e n c e ( ~Having j. bound to a signal sequence the role of the SRP is two fold: 1) to maintain the nascent chain in a translocation competent ( i t . unfolded) state compatible with membrane insertion(lO,ll);2) to tag the nascent chain as destined for the ER. Upon arrival of the nascent chain/ribosome/SRP complex at the ER the signal sequence is released from SRP54 in a mechanism which requires both the docking protein complex (a and p subunits) and GTP(12,13).While the release of the nascent chain does not require GTP hydrolysis the subsequent release of the SRP from the docking protein (Fig. 3 ) .
-
N
signal-anchor sequence SRP: signal ncogniiion panicle DF: docking prolein TI & T2: components of trmslmlion machinery.
Fig. 3. SRP mediated ER targeting cycle. SRP binds to the signal sequence of the growing nascent chain and targets the nascent chainlrihosomelSRP complex, to the ER membrane. Upon interaction with the docking protein complex, the nascent chain is released from SRP and interacts with the translocation machinery. SRP is released from DP upon GTP hydrolysis and rejoins the cytosolic pool. The synthesis of the nascent chain is completed at the membrane and the protein is stably inserted into the lipid bilayer.
Membrane insertion After the release of the nascent chain from SRP, it interacts with the ER translocation machinery. The nascent chain now has two possible fates (Fig. 2): 1) it can be completely translocated across the ER membrane and enter the lumen as a secretory protein('.l5); 2) a selective translocation of only part of the protein can occur leaving the other region cytosolically exposed and the protein stably inserted in the membrane. Thus during membrane insertion a partial rather than a complete translocation of the nascent chain occur^(^^.^^)). The translocation event itself i s probably identical in both cases and the type of signal sequence present will determine the fate of the protein after translocation has been initiatedcs,17) (Fig. 2). The final orientation which a membrane protein assumes in the lipid bilayer is dependent upon the kind of signal sequence it p o s s e s ~ e s ( ~Proteins ~ ~ ) . which span the membrane once and have a cleavable signal sequence always adopt a type I orientation, the NH2 terminus being extracytoplasmic (Fig. 2). Signal-anchor proteins which span the membrane once can adopt either a type I or a type I1 oricntation (Fig. 2). The orientation of signal-anchor proteins is determined b y the properties of the nascent chain and in particular the number of charged residues present in the two hydrophilic regions adjacent to the hydrophobic core of the signal-anchor s e q u e n ~ e ( ~ ~On ~ ~ ' the ~ ) . basis of theoretical consideration~('9,*~), and supporting experimental a mechanism where thc nascent chain inserts into the membrane in a loop conformation (e.g. Fig. 3) is currently f a v o ~ r e d17). (~~~~
The Generation of Translocation Intermediates The environment of the nascent chain during membrane insertion has been investigated by several approaches. A prerequisite of these investigations has been the ability to trap the nascent chain during the process of membrane insertion and generate 'translocation intermediates'. The underlying assumption is that the interactions of these translocation intermediates with components of the ER are the same as those made by the nascent chain during the normal course of membrane insertion. Such translocation intermediates can be made by using an artificially truncated mRNA to prime the in vitro synthesis of the nascent chain under study(22).Since the tmncated inRNAs lack a stop codon the ribosome stalls at the 3' end of the mRNA and thc resulting incomplete nascent chain remains stably associated with the ribosome. If translation is carried out in the presence of SRP and rough microsomes (ER vesicles) the protein is correctly targeted to and inserted into the microsomal membranc. Howcver, since thc COOH-terminus of the nascent chain remains attached to the ribosome, the nascent chain is jammed in the translocation site(23)(Fig. 4).
Cross-Iinking A direct method to analyse the immediate environment of these translocation intermediates has proved to be cross-link-
Table 1. Summary of ER proteins which are crosslinked to ing. Since the nascent chains can be made in vitro they can trunslocation intermediules easily be radioactively labelled. If the labelled nascent chain is then covalently cross-linked to a second, unlabelled, procrosslinked to proteins with: tein (Fig. 4), the resulting cross-linking product can be detected by the appearance of a radioactive product which is cleavable Signal-anchor M.wt (ma) signal sequence sequence Reference larger than the original nascent chain. Since the nascent chain is trapped during the act of translocation it is reasonable to Non-glycoproteins believe that proteins which can be cross-linked to these 125I imp34 34 yes nd P37 37 Yes yes [26]& I translocation intermediates are candidates for components of the translocation machinery. GIycoproteins SSRa 35 Yes yes [26,41] Two methods have been used to cross-link translocation TRAM 36 ycs nd I1 intermediates to adjacent ER proteins. In the first ‘phomp39 39 Yes yes [23,27] tocross-linking’ approach, a modified lysine residue is incorporated into the nascent chain during in vitro synthesis. Upon nd=not determined ultraviolet irradiation the side chain of the modified lysine I=S. High unpublished data II=T.A. Kapoport pers. comm. forms a highly reactive radical which will rapidly react with NB. mp39 is equivalent to SSRa and TRAM. imp34 and P37 are nearby proteins(24). A second approach has been to use probably the same coinponent. bifunctional cross-linking reagents, which react specifically only with certain amino acid side chains, and add these to the translocation intermediates generated as d e ~ c r i b e d ( ~ In ~.~~). tors. These include the nascent chain used, the length of the both cases only a very small number of cross-linked products nascent chain in the translocation intermediate and the crossare observed consistent with only a small subpopulation of linking method. We have recently found that the exact posiER proteins being in the immediate vicinity of the nascent tion within the nascent chain from which cross-linking chain translocation intermediate. occurs also influences the ER protein which is crosr-linked The specificity of this interaction between the nascent (S. High, B. Martoglio, J. Brunner and B. Dobberstein, chain and ER proteins has been tested by determining the unpublished data). Thus, different regions of the nascent cross-linking partners of the nascent chain after the translochain of a translocation intermediate are in contact with difcation intermediate has been disrupted by removal of the ferent ER proteins. To date cross-linking has identified 34 ribosome. This can be accomplished by treatment of the riboand 37 lcDa non-glycoproteins and at least two 35-36 kDa some with EDTA or puromycid2@,or by allowing the synglycoproteins as candidates for components of the translocathesis of the complete protein rather than a truncated vertion machinery (see Table I). So far only a 36 kDa glycoproion(^^). In both cases, although the nascent chain clearly tein denoted ‘TRAM’ has been shown to play a direct role in remains within the membrane, it is no longer found crosstranslocation (sce below). linked to the proteins observed when the translocation intermediates are used. Thus, the proximity of the nascent chain to these ER components is transient and dependent upon a Indirect Analysis ribosome-associated translocation intermediate. The biochemical properties of translocation intermediates The major proteins which have been identified by crosshave also been examined in an attempt to determine their linking approacheb are shown in Table 1. The pattern of ER environment. Translocation intermediates of the secreted proteins which is cross-linked is dependent upon several facprotein preprolactin have been found to be extracted from the membrane by agents such as urea. This suggests that at this stage the nascent chain may not be completely integrated into the lipid bilayer but rather in an aqueous environment or in contact with other proteins(28).When translocation intermediates are solubilised at high detergent concentrations, the nascent chain is significantly protected from added protease, including NHz-terminal regions exposed outside the riboCytosol some. This suggests that after solubilisation of the membrane the protein components of the translocation site stay associated with the membrane inserted region of the nascent chain Membrane and protect it from the added protease(2Y).
Lumen Fig. 4. A ribosome bound translocation intermediate.
* indicates a
residue of the nascent chain suitable for cross-linking to adjacent components. The hatched column represents an ER protein in proximity to the translocation intermediate.
Insertion into the ER Requires Protein Components A direct role for ER proteins in mediating membrane insertion and translocation is indicated by the observation that some covalent modifications of ER component{ can com-
pletcly block these processes. It has been shown that an NEM sensitive protein component is required for correct translocation in ~ i t r d ~This ~ ) component . is clearly distinct from the docking protein which is also sensitive to NEM and is necessary for correct targeting (see above) - a prerequisite for membrane insertion. Recently it has been found that the covalent modification of ER membrane components with the ATP analogue 8-azido ATP also blocks both the translocation of secreted p r o t e i n ~ ( ~and ' , ~ the ~ J insertion of membrane proteins (S. High, unpublished data). It is not presently known which components of the translocation machinery are sensitive to these modifications.
Genetic Analysis A genetic \election has been used with the yeast Sacchnmmyces cerivisiae to allow the identification of genes which encode proteins that are required for the translocation of proteins across the membrane of the ER(33.34).Three integral membrane proteins of the ER were identified: Sec6lp, Sec62p and Sec63p. When these proteins are absent or defective the efficiency of translocation of secretory proteins acros~the ER is much r ~ d u c e d ( ~ ? -Sec6lp, '~) Sec62p and Sec63p are found as part of a complex of proteins present in the Saccharomyces cerevisiae ER(36Jand the evidence that this complex forms at least a part of the membrane insertion machinery is compelling. While the suspicion is strong(?'), there is as yet no direct evidence that proteins homologous to Secblp, Sec62p and Sec63p are present in the mammalian ER.
is not essential for membrane insertion or tr'anslocation in vitrdS2).When antibodies against the other ER components identified by cross-linking are available, such reconstitution techniques will provide a useful method to test their role in membrane insertion and translocation.
Accessory Proteins In addition to proteins which are directly involved in membrane insertion and translocation, other proteins appear to be close to the site of translocation but not directly involved in the process per se. Antibodies directed against ribophorin I and SSRa have been shown to block the translocation activity of membrane^(^^,^^). Ribophorins I and I1 form a part of the complex responsible for the addition of asparaginelinked carbohydrate to nascent chains emerging on the lumenal side of the ER(44).SSRa has recently been shown to be a major calcium binding protein of the ER and its function is at present unclear.
The Machinery for Membrane Insertion There is now a wealth of data to show that ER proteins are required to mediate the insertion of nascent chains into the lipid bilayer. Current evidence also suggests that both membrane insertion and translocation use the same ER machinery. A number of possible types of translocation site across the lipid bilayer of the ER have been proposed: 1) A protein and lipid mediated translocation site(5).The hydrophilic regions of the protein which are translocated across the membrane would contact ER proteins while the hydrophobic signal sequence would interact directly with lipids. Reconstitution 2) A transient protein-lined translocation ~ i t e ( ~ , ' ~The ,l~). formation of the translocation site would be coupled to the In mammalian cells biochemical techniques have provided a arrival of a correctly targcted nascent chain complex at the way in which the role of an ER protein in membrane insertion ER. The translocated region of the protein would pass and translocation can be tested directly. It has been shown through the channel while the hydrophobic signal would be that the ER vesicles (rough microsomes) which are used to retained. study membrane insertion in vitro can be taken apart and subsequently put back together or r e c o n ~ t i t u t e d ( ~ ~The - ~ ~ ) . 3) A gated protein-conducting channel(46).The opening of the chaiiiiel would be closely regulated to ensure that only the reconstituted vesicles are capable of correctly translocating translocated portion of a nascent chain can normally pass secreted protcins into their l ~ m e n ( ~ * and - ~ ~of ) , correctly inserting membrane proteins into their lipid b i l a ~ e r ( ~The ~ ) . through. During the course of membrane protein insertion it is clear requiremcnt for any ER component in the translocation that the hydrophobic signal-anchor or stop transfer sequence process can thus be tested by removing it from the solubilised must exit the translocation site in the lateral plane and enter membrane fraction prior to reforming the vesicles. the lipid bilayer at some stage. Cleavable signal sequences If a lectin column is used to deplete the solubilised ER of may also exit the translocation site prior to cleavage by signal all glycoprotcins the resulting reconstituted membrane vesipeptidase although this is at present not known. The exit of a cles show a much reduced translocation activity("). It has hydrophobic signal raises two questions: how and when? recently been shown that the addition of a single purified glyHow the interaction occurs is straightforward to explain for a coprotein to the depleted mixture can restore the ability of the protein/lipid translocation site since lipid is already in close reconstituted vesicles to translocate certain secreted proteins. contact with the signal. In the case of a transient protein-lined The 36 kDa glycoprotein responsible for restoring the in site, its very nature would allow for a subunit to be displaced vitro translocation activity to the depleted vesicles has been at some point giving the nascent chain lateral access to the named the translocating chain-associating membrane prolipid bilayer('@. A protein-conducting channel would also tein(TRAM) (T.A. Rapoport, pers c o r n ) . Reconstitution have to allow lateral access to the lipid bilayer in order to has also been used to show that a previously identified 35 allow the stable integration of membrane proteins'46). kDa glycoprotein, named the signal sequence receptor If membrane insertion does occur initially in an all protein (SSRCX)(~]), although in proximity to some nascent chains environment, be it of a transient or a permanent nature, then during their membrane insertion and tran~location(~~,~~,~~,~l),
how is the message that the hydrophobic signal sequence should now enter the lipid bilayer conveyed? One possibility is that this is linked to the departure of the ribosome from the ER membrane upon the completion of protein synthesis. A similar mechanism has been proposed to act as a signal for closing a gated protein-conducting channel upon the completion of translocation("). Coupling the lateral exit of the hydrophobic sequence of a membrane protein from the translocation site to the departure of the ribosome is also consistent with cross-linking data. Thus, in the absence of the ribosome the previously identified contacts betwecn nascent membrane proteins and ER proteins are no longer o b ~ e r v e d ( ~ ~The 3 ~release ~). of the ribosome would allow a transient translocation machinery to dissociate, enabling the signal-anchor or stop-transfer sequence of a membrane protein to enter the lipid b i l a ~ e r ( ~., l ~ ) The evidence for the involvement of ER proteins in membrane insertion has already been discussed. None of these data, however, approach the question of what the physical make up of the translocation site looks like. One approach to this problem has been to use electrophysiological techniques to analyse the channels present in the ER. Using this approach, data consistent with the translocation site being a gated channel have been obtained'46).
The Membrane Insertion Site - a Model The work I have described leads me to make the following general conclusions about the machinery responsible for membrane insertion into the ER (see Fig. 5): 1) it is a complex formed from a number of heterologous protein subunits; 2) 'core' proteins of the complex are directly involved in the
process of membrane insertion and are absolutely required for function; 3) a number of accessory proteins are closely associated with the complex but do not play a direct role in membrane insertion. Rathcr, they are involved in related functions which normally occur during the membrane insertion process. Membrane insertion or translocation is only one step in a complicated journey which the protein makes. This includes targeting to the ER, insertion into the membrane or complete translocation across it into the lumen, and then subsequent sorting to the correct subcellular destination (see Fig. 1). The targeting machinery must be either closely associated with the translocation site or a part of it. Thus, after the release of the nascent chain from SRP54, it is directly transferred to ER membrane proteins(13).After insertion, the nascent chain emerges on the luminal side of the membrane. lf a suitable cleavage site is present, the NH2-terminal signal will be cleaved from the nascent ~ h a i n ( l $ ~ Both . ~ > ~secreted ). and membrane proteins are quickly modified by the addition of carbohydrate to suitable asparagine residuescs7). In addition the formation of disulphide bonds occurs rapidly implying that the catalytic activity of protein disulphide isomerase is close at hand(48'. Chaperones such as BiP may bind to the emerging nascent chain on the luminal side of the ER and mediate the correct folding, and assembly (in the case of oligomcric proteins), of the luminal portion of nascent membrane proteins(49).In yeast, there is evidence to suggest that BiP may also be directly required for efficient translocation to occur(S0,").
Conclusions The insertion of proteins into the membranc of the ER is mediated by a proteinaceous machinery. This translocation machinery consists of a protcin complex made up of scvcral heterologous subunits and is responsible both for the insertion of membrane proteins into the lipid bilayer and the complcte translocation of sccretory proteins into the lumen of the ER. While the specific nature of the proteins which make up the translocation machinery of mammalian cells remains unclear, several good candidates have now been identified. A detailed structure of the translocation complex and the mechanism by which translocation occurs would secm to be within reach.
I
pD1
J
Fig. 5. Model translocation site. The membrane bound ribosome is synthesizing a type I1 signal-anchor membrane protein. The hydrophobic core of the signal-anchor sequence is shaded black. DP = docking protein. T1 and T2 = protein components of the translocation machinery. SPasc = signal peptidase. CHO = oligosaccharyltransferase complex, of which ribophorins I and 11 form a part(44)). RR = putative ribosome SSR = signal sequence receptor (asubunit). BiP = heavy chain binding protein. PDI = protein disulphide isomerase.
Acknowledgements Many thanks to Reid Gilmore and Tom Rapoport for communicating results prior to publication. Thanks also to Sgren Andersen, Bernhard Dobberstein, Jonathan Howard, Joen Luirink, Henrich Lutcke and Jean Pieters for their careful reading of the manuscript and useful suggestions for its improvement.
Note added in proof The work on thc TRAM protein is now published, see Gorlich, D., Hartmann, E., Prehn, S. and Rapoport, T. A. (1992). Nature 357,47-52.
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Stephen High is at thc European Molccular Biology Laboratory. Meycrhofstrasse 1. Postfach 102209,6900 Hcidclbcrg, Germany.
