The EMBO Journal vol. 13 no. 17 pp.3973 - 3982, 1994

Systematic probing of the environment of atranslocating secretory protein during translocationthrough the ER membrane

Walther Mothes, Siegfried Prehn1 andTom A.Rapoport2Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse10, 13125 Berlin-Buch and lInstitute for Biochemistry, Humboldt-University Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany

2Corresponding author

Communicated by T.A.Rapoport

We have extended a previously developed photo-crosslinking approach to systematically probe theprotein environment of the secretory protein prepro-lactin, trapped during its transfer through the endo-plasmic reticulum membrane. Single photoreactivegroups were placed at various positions of nascentpolypeptide chains of various length, corresponding todifferent stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In allcases, the polypeptide segment extending from theribosome was found to be located in a membraneenvironment that is formed almost exclusively fromSec6l1c, the multi-spanning subunit of the Sec6lpcomplex that is essential for translocation. At earlystages of the translocation process, before cleavage ofthe signal sequence, almost the entire nascent chainemerged from the ribosome contacts Sec6la. The'translocating chain-associating membrane' proteininteracts mainly with the region of the signal sequencepreceding its hydrophobic core. Our results suggestthat the nascent chain is transferred directly from theribosome into a protein-conducting channel, the majorconstituent of which is Sec6la.Key words: endoplasmic reticulum/photocrosslinking/protein translocation/Sec6l/translocating chain-associat-ing membrane protein

IntroductionProtein transport across the endoplasmic reticulum (ER)membrane is initiated in general by an interaction of thesignal sequence of a growing nascent polypeptide with the54 kDa polypeptide component of the signal recognitionparticle (SRP54) [for a review see Rapoport (1992)].Upon binding of the entire complex of ribosome, nascentchain and the SRP to the SRP receptor (docking protein)of the ER membrane, the ribosome becomes membrane-bound and the nascent polypeptide chain is transferredinto the membrane. For secretory proteins and manymembrane proteins it is assumed that the nascent chain isinserted in a loop structure into the membrane, with theN-terminus remaining in the cytoplasm and with one partof the hairpin being the signal sequence. The subsequent

movement of the C-terminal portion of the hairpin ispostulated to occur through a hydrophilic protein-con-ducting channel that is formed at least in part fromtransmembrane proteins (Blobel and Dobberstein, 1975).The idea of a channel is supported by electrophysiologicaldata that indicate the occurrence of large ion-conductingchannels following the release of the nascent chains frommembrane-bound ribosomes (Simon and Blobel, 1991).The inside of the channel may have a hydrophilic environ-ment, as determined by measurement of the fluorescentlife-time of probes incorporated into translocating poly-peptide chains (Crowley et al., 1993).The translocation apparatus of the ER membrane seems

to be surprisingly simple; in some cases only two proteincomponents are required for the translocation of poly-peptides into reconstituted proteoliposomes (Gorlich andRapoport, 1993): (i) the SRP receptor (two subunits;Tajima et al., 1986), which is probably required only forthe targeting of a nascent chain to the ER but not for itsactual transfer through the membrane; and (ii) the Sec6lpcomplex (three subunits). The a-subunit of the Sec6lpcomplex is a good candidate to be a major component ofthe postulated protein-conducting channel. It probablyspans the membrane 10 times and has in these putativemembrane-spanning segments several hydrophilic andcharged amino acids that could contribute to the formationof a hydrophilic channel (Gorlich et al., 1992b). Trans-locating polypeptides can be crosslinked to Sec6la bothin yeast and mammalian microsomes at different stagesof their membrane passage (Gorlich et al., 1992b; Muschet al., 1992; Sanders et al., 1992; High et al., 1993a).The translocation of most polypeptides also requires the

presence of a third membrane component, the translocatingchain associating membrane (TRAM) protein (Gorlichet al., 1992a; Gorlich and Rapoport, 1993). The TRAMprotein may be in contact with nascent chains only atearly phases of their transfer through the membranebefore the signal sequence has been cleaved off (Gorlichet al., 1992a).

Although the previous crosslinking experiments haveshown that Sec6la and the TRAM protein are closeto polypeptides which are in transit through the ERmembrane, the exact molecular environment that poly-peptides meet during their passage through the membraneis unknown. So far, in most experiments several sites ofthe polypeptide chain could be involved in crosslinking.Photocrosslinking experiments, with photoreactive lysinederivatives incorporated into nascent chains, generallyemployed translocation substrates which contained severallysine residues that could carry the probes. Crosslinkingwith bifunctional chemical reagents (Gorlich et al., 1990,1992a; Kellaris et al., 1991) also lacked specificity withrespect to the interacting site in the translocating poly-peptide. It is therefore not even certain that it is the

© Oxford University Press 3973

W.Mothes, S.Prehn and TA.Rapoport

membrane-inserted region of a translocating polypeptidethat contacts Sec6la or the TRAM protein.To overcome these problems it would be desirable to

position precisely photoreactive chemical groups in atranslocating polypeptide chain. Two approaches may beconsidered. (i) One may use the 'classic' method,employing photoreactive lysine derivatives incorporatedinto a nascent polypeptide chain synthesized in vitro inthe presence of modified lysyl-tRNA (Krieg et al., 1986;Kurzchalia et al., 1986) under conditions in which onlyone lysine codon is present in the mRNA. (ii) The probesmay be incorporated at sites defined by stop codonsin the mRNA, which are subsequently suppressed withsuppressor-tRNA charged with a photoreactive amino acidderivative. The feasibility of the second approach hasalready been demonstrated by the incorporation of photo-reactive phenylalanine derivatives at a few positions ofshort preprolactin chains of 86 amino acids (High et al.,1993b). The results indicated that the TRAM proteininteracts with amino acid residues preceding the hydro-phobic core of the signal sequence, whereas Sec6lainteracts primarily with the core and with residuessucceeding it. Further application of this method awaitsthe more general availability of the reagent. Also, thereis some uncertainty as to whether stop codons can besuppressed with the same efficiency at every position ofthe polypeptide chain.

Using the 'classic' photocrosslinking approach, we havenow carried out a systematic analysis of the membraneprotein environment of preprolactin chains which weretrapped during their transfer through the ER membrane.Single lysine codons were placed in the mRNA at selectedsites so that photocrosslinking could only occur fromknown positions of the polypeptide chain. Employingdifferent translocation intermediates, it could be demon-strated that the region of the polypeptide chain precedingthe ribosome-buried portion gives almost exclusivelycrosslinks to Sec6la, suggesting that the latter forms aprotein-conducting channel. Our results also suggest thatthe nascent chain is inserted initially into the membranein a loop structure in which the N-terminal part of thesignal sequence contacts the TRAM protein, whereas therest of the membrane-incorporated chain is in proximityto Sec6lac. Additional membrane proteins seem to contactthe nascent polypeptide chain at the lumenal side of themembrane.

ResultsExperimental strategyTo position precisely photoreactive lysine derivatives inpreprolactin polypeptide chains trapped during transloca-tion, we first removed all lysines of the wild type protein,up to position 154, yielding a 'lysine null' mutant (N)that is not expected to incorporate photoreactive groups(Figure lA). Subsequently, single lysine codons wereintroduced at various selected positions by site-directedmutagenesis (schemes in Figure lB and C). Thus, thephotoreactive probes could be placed in a systematicmanner into the nascent chain, starting with positionsexpected to be in the ribosome up to locations in thelumen of the ER membrane.

In initial experiments, a late stage of translocation was

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Fig. 1. Schematic illustration of the systematic crosslinking approach.(A) To position precisely photoreactive lysine derivatives, wild typepreprolactin containing lysines at the indicated positions wasmutagenized to result in a 'null' mutant lacking all lysines up toposition 154. Subsequently, single lysine codons were introduced atvarious positions, as indicated schematically in (B) and (C). (B) A latetranslocation intermediate was produced by in vitro translation oftruncated mRNAs coding for 169 or 187 amino acids (169mer or187mer) in the presence of ER membranes. The N-terminus of thepolypeptide chain is transferred across the membrane, whereas theC-terminus remains in the cytoplasm attached to the ribosome.Photoreactive lysine derivatives were incorporated into the polypeptidechain at the position defined by the single lysine codon (dots) andcrosslinks to membrane proteins were analyzed. (C) An earlytranslocation intermediate was produced by translation of truncatedmRNAs coding for 86 or 132 amino acids (86mer or 132mer). In thiscase, the signal sequence (boxed) is not yet cleaved and the nascentchain presumably adopts a loop structure.

studied in which the signal sequence has been cleaved offand in which the translocating nascent chain has adopteda trans-membrane orientation (Figure 1B). To this end,truncated mRNAs coding for fragments of preprolactincontaining 169 or 187 amino acids (169mer or 187mer)were translated in vitro in the presence of microsomalvesicles, [35S]methionine and modified lysyl-tRNA (seeWiedmann et al., 1987). Under these conditions theribosome migrates up to the end of the mRNA, but cannotfall off because of the lack of a stop codon. The N-terminusof the synthesized polypeptide fragment is translocated intothe lumen of a microsomal vesicle, whereas the C-terminusremains in the cytoplasm attached to the ribosome aspeptidyl-tRNA. The polypeptide is radioactively labeledand contains a photoreactive lysine derivative at the

3974

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Fig. 2. Reconstructing the crosslinking pattern of wild type 169mer from that of single-lysine mutants. The crosslinking pattern of the wild type169mer of preprolactin (wt) is compared with that of mutants containing either none (N) or only single lysine codons (positions indicated bynumbers above the lanes). Translocation intermediates containing photoreactive groups at all lysine positions were produced and irradiated (lanes 7-18). Control samples were not irradiated (lanes 1-6). The membrane protein crosslinks were analyzed either directly by SDS-PAGE andfluorography (total products) or first immunoprecipitated with antibodies against Sec6la (anti-Sec6la). The arrow heads indicate the positions ofcrosslinks to membrane proteins other than Sec61a. 169mer +SP and -SP, 169mer with and without signal peptide, respectively.

position defined by the single lysine codon. Upon irradi-ation, a carbene is produced that reacts rapidly withneighboring molecules. Crosslinks to membrane proteinscan be identified following alkaline extraction of themembranes.To study earlier phases of translocation, shorter trun-

cated mRNAs were employed, coding for preprolactinfragments of 86 or 132 amino acids (86mer or 132mer).In these cases, the signal sequence is not yet cleaved off(Figure IC).

Reconstructing the crosslinking pattern of the wildtype proteinTo test the validity of our approach, we first comparedthe crosslinking pattern of the wild type 169mer, containingseven lysine codons, with that of mutants containing onlysingle lysine codons (Figure 2). The pattern obtained withthe wild type is rather heterogeneous (Figure 2, lane 7),as noted before (Gorlich et al., 1992b). However, theheterogeneity is more pronounced than in the previousexperiments in which the crosslinking reagent was incorp-orated only in C-terminal lysine positions. The majorcrosslinks with the wild type polypeptide occur to theSec6la protein (Figure 2, lane 13). Several other crosslinkswere seen among the total products (lane 7), includingcrosslinks to proteins of -70, 30 and 27 kDa [indicated byarrow heads; sizes estimated from those of the crosslinkedproducts after subtraction of the size of the signal peptide-cleaved form of the 169mer (-15 kDa)]. As expected, thelysine-null mutant (N) did not give any crosslinks tomembrane proteins (lanes 12 and 18). Each of the singlelysine mutants, however, produced a characteristiccrosslinking pattern corresponding to a subset of productsseen with the wild type protein (lanes 8-11). Strongcrosslinks to Sec6la were observed with the probes atpositions 136 or 99, and weaker ones with those atpositions 78 or 72 (lanes 14-17). Interestingly, dependingon the position of the crosslinker, the Sec6la crosslinks

had different mobilities in SDS gels (apparent molecularweights 43-53 kDa) and in the case of position 136 gavetwo bands (e.g. lane 14). The latter may be caused bycrosslinking from a single site of the nascent chain todifferent regions of the Sec61a molecule, resulting inproducts of different electrophoretic mobilities. However,it cannot be excluded that they are produced by the signalsequence-containing and -lacking forms of the 169mer,respectively, even though the latter was in great excess.In any case, the data explain the heterogeneity of theSec6la crosslinking pattern seen with the wild type. Thecrosslinking to the other membrane proteins could also befully accounted for by the different positions of the probes:the crosslinks to the 70 and 27 kDa proteins were producedexclusively by the mutant with the crosslinker at position72 (lane 11), and that to the 30 kDa protein by the mutantwith the probe at position 78 (lane 10). These resultsindicate that the complex crosslinking pattern of thewild type protein can be reconstructed from that ofthe individual single-lysine mutants and that differentmembrane proteins contact the nascent chain at differentpositions.

Systematic crosslinking with the 169merNext, we positioned single crosslinking probes in a more

systematic manner into the 169mer between amino acids31 and 151 (Figure 3). Crosslinks to integral membraneproteins were only observed with probes positionedbetween amino acids 72 and 139 (Figure 3A, lower panel).Most likely, the positions 143-169 are buried inside theribosome and therefore cannot contact membrane proteins.The region of the polypeptide chain between positions 31(signal peptide cleavage site) and 63 is probably locatedfar enough in the lumen of the ER not to give crosslinksto integral membrane proteins.The crosslinking pattern with probes in the region

preceding the ribosome-buried portion (positions 99-139)was found to be surprisingly simple, with major bands

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ml-Fig. 3. Systematic crosslinking with single-lysine mutants of the 169mer. (A) Translocation intermediates were produced with mutants of the 169merof preprolactin, containing single lysines at the indicated positions (numbers above the lanes) which carry the crosslinking probes. After irradiation,crosslinks to membrane proteins were analyzed by SDS-PAGE and fluorography (total products). For two regions of the polypeptide chain, lysinecodons were placed at every single position (upper panels). The arrow and arrow heads indicate the positions of crosslinked products containingmembrane proteins other than Sec6la. (B) Parallel samples of the experiment described in (A) were submitted to immunoprecipitation withantibodies against Sec6la. The precipitated material was subsequently analyzed by SDS-PAGE and fluorography.

produced by a crosslinking partner of -43-53 kDa (Figure3A). Minor crosslinks to a small partner of -7-8 kDa(indicated by an arrow) were also observed with some ofthe mutants. Immunoprecipitation experiments demon-strated that the large crosslinking partner is Sec6la (Figure3B, lower panel). The efficiency of immunoprecipitationof the crosslinked products was generally -40%, similar tothe efficiency of immunoprecipitation of non-crosslinkedSec6la (data not shown). None of these crosslinks boundto concanavalin A (data not shown), indicating that theslightly smaller glycosylated TRAM protein is not amongthe crosslinking partners. As noted before, different posi-tions of the nascent chain appear to contact differentregions of the Sec6la molecule since the crosslinkedproducts have different electrophoretic mobilities. In addi-tion, each of the mutants with lysines between positions131 and 139 gave two bands of Sec6la crosslinks.

In our initial screens the lysine codons were placedat every fourth position within the region of positions 99-139. To test whether in this region Sec6la contacts eachamino acid residue of the nascent polypeptide, we placedlysine codons in two regions (111-118 and 131-136) atevery single position. For both regions all the mutantsgave mainly crosslinks to Sec6la (Figure 3A and B,upper panels). Thus, it seems that for every position ofthe region of 40 amino acids preceding the ribosome-buried portion of the nascent chain, Sec6la is the majorcrosslinking partner (see scheme in Figure 7).

It should be noted that some positions within the region99-139 gave reproducibly weaker crosslinks (e.g. positions107, 111, 116 and 123). This may indicate that thesepositions are not in close contact with Sec61a, that suitablechemical groups are not available for interaction with thecarbene, which is not entirely unspecific in its reactions(Brunner and Richards, 1980), or that the reaction of thecarbene is quenched, for example by neighboring wateror lipid molecules. A more pronounced positional effectthan for Sec6la was observed for the unidentifiedcrosslinks to the small protein; for example, they wereseen for positions 118 and 113 (see arrows) but not forpositions in between.The nature of the crosslinks seen with mutants carrying

photoreactive probes in positions 72 or 78 (see arrowheads) is unknown as yet. A portion of the smallest ofthese membrane protein crosslinks could be immunopre-cipitated by antibodies against TRAPa (previously calledSSRa) (data not shown). The 70 kDa protein, crosslinkedfrom position 72, is a glycosylated protein (not shown).

Crosslinking with the 187merTo test whether the results obtained with the 169mer canbe generalized, we have employed a somewhat longerfragment of preprolactin (l87mer). If the crosslinks dependsolely on the position of the crosslinker in the nascentchain relative to the membrane, one would expect a shiftof 18 residues (187-169) in the crosslinking pattern.

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Fig. 4. Crosslinking with single-lysine mutants of the 187mer.Translocation intermediates were produced with mutants of the 187merof preprolactin, containing photoreactive lysine derivatives at theindicated positions (numbers above the lanes). After irradiation,crosslinked products were analyzed by binding to a Ni columnfollowed by either SDS-PAGE and fluorography (total products; A)or by immunoprecipitation with antibodies against Sec6la andphosphorimaging (anti-Sec6la; B). The two small triangles in(A) indicate the position of peptidyl-tRNA which was incompletelyremoved in the puromycin reaction. The product of -69 kDa (A)apparently also contains Sec6la (see B).

The experiments with longer nascent chains turned outto be more difficult than anticipated. Translation of thecorresponding truncated mRNA resulted in a number ofnon-completed nascent chains which also gave crosslinksto membrane proteins. To analyze exclusively thecrosslinks of completed polypeptides, we therefore con-

structed an mRNA coding for a 187mer with a C-terminalhistidine tag (see Materials and methods). This tag allowedthe purification of the crosslinked products of thecompleted 187mer by binding to a Ni column.

Crosslinks of the 187mer to membrane proteins were

observed with mutants carrying probes in positions from-115 to 151 (Figure 4A) and, as for the 169mer, allthe major products contained Sec6la (Figure 4B). Theobserved crosslinking region corresponds to a shift of -16residues compared with the 169mer (115-99) (no positionbeyond 151 was tested, but presumably a few more

C-terminal residues of the 187mer are located in themembrane). The region of crosslinking to Sec6la (Figure4B) had less defined borders when compared with that ofthe 169mer, probably because the longer nascent chainshowed more fluctuation in its structure. Nevertheless,comparison of the results obtained with the two latetranslocation intermediates indicates that the crosslinkingpattern is largely determined by the spatial position of theprobe relative to the membrane.

Fig. 5. Crosslinking with single-lysine mutants of the 132mer.Translocation intermediates were produced with mutants of the 132merof preprolactin, containing photoreactive lysine derivatives at theindicated positions (numbers above the lanes). After irradiation,crosslinks to membrane proteins were analyzed either directly bySDS-PAGE and fluorography (total products; A) or afterimmunoprecipitation with antibodies against Sec6la (anti-Sec6la; B)or against the TRAM protein (anti-TRAM; C). The arrow indicates theposition of a crosslinked product containing an unidentified 120 kDaprotein which was sometimes observed. The doublet of the 132mer isdue to signal peptide cleavage induced by puromycin treatment aftercrosslinking.

Crosslinking with early translocation intermediatesNext, we investigated the crosslinking pattern of nascentchains from which the signal sequence is not yet cleavedoff, employing shorter fragments of preprolactin con-taining 132 or 86 amino acids. Crosslinking with singlelysine mutants of the 132mer indicates that most positionscontact membrane proteins, with the exception of the last-30 residues (beyond position 103) which seem to beburied inside the ribosome (Figure 5A). Sec6la was themajor crosslinking partner for all mutants carrying lysineswithin the range from position 18 to 99 with the exceptionof position 63 (Figure 5B). The range in which Sec6lacrosslinks were observed is clearly much longer than atlater stages of translocation (-80 residues for the 132merversus 40 for the 169mer). The weak crosslinks to a

protein of -120 kDa (marked by an arrow) have not beenidentified and were only seen in some experiments withthe 132mer.The major crosslinked product seen with the probes

present in positions 4 and 9 (only the double lysine mutantwas tested) could be immunoprecipitated with antibodiesagainst the TRAM protein (Figure 5C). A minor TRAMcrosslink was also observed with the mutant carrying theprobe at position 31 (highlighted by a dot), but all otherpositions did not give crosslinks with this protein (FigureSC and data not shown).

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Fig. 6. Crosslinking with single-lysine mutants of the 86mer.Translocation intermediates were produced with mutants of the 86merof preprolactin, containing photoreactive lysine derivatives at theindicated positions (numbers above the lanes). After irradiation,crosslinks to membrane proteins were analyzed either directly bySDS-PAGE and fluorography (total products; A) or afterimmunoprecipitation with antibodies against Sec6la (anti-Sec6la; B)or against the TRAM protein (anti-TRAM; C). The position of acrosslinked product containing a 7-8 kDa protein is indicated by anarrow. The doublet of the 86mer is due to signal peptide cleavageinduced by puromycin treatment after crosslinking.

Results similar to those with the 132mer were obtainedwith the 86mer (Figure 6). Positions 31 and 47 gavestrong crosslinks to Sec6la and position 18 weak ones,presumably because this mutant was only poorly trans-located (Figure 6B). Crosslinking to Sec6l1a from position18 was stronger in previous experiments in which adifferent photoreactive group was incorporated (Highet al., 1993b). Only the positions 4 and 9 were found tocontact the TRAM protein (Figure 6C), in agreement withprevious data (High et al., 1993b). The last 30 residues(55-86) again seem to be buried inside the ribosome. Theminor crosslinks from this region are probably to ribosomalproteins which were incompletely removed by the extrac-tion with alkali.

DiscussionWe have extended our previously employed photo-crosslinking approach to probe the membrane environment

of the secretory protein preprolactin trapped during itstransfer through the ER membrane. Single photoreactivegroups were placed in a systematic manner at variouspositions into polypeptide chains of various length, corres-ponding to different stages of the transport process.Subsequently, crosslinking was induced by irradiation,and crosslinked products containing membrane proteinswere analyzed. Our results are summarized schematicallyin Figure 7 and lead to a model showing the environmentof a secretory polypeptide during its transfer through theER membrane (Figure 8).About 30 C-terminal residues do not give crosslinks to

membrane proteins with all chain lengths, most probablybecause they are buried inside the ribosome. A similarestimate was obtained when the crosslinking of nascentchains to SRP was analyzed in the absence of membranes:the C-terminal 32 residues did not give crosslinks to SRP54(B.Misselwitz, W.Mothes and T.A.Rapoport, unpublishedresults). Our estimates on the length of the ribosome-buried polypeptide segment are somewhat lower thanthose obtained by analyzing the size of the polypeptidethat is protected from proteolytic attack (-35-40 residues)(Malkin and Rich, 1967; Blobel and Sabatini, 1970). Onepossible explanation is that membrane proteins and SRPmay project into the ribosome and thereby shorten thelength of the actual ribosome channel.The region of the nascent chain preceding the one

buried inside the ribosome was found to be in themembrane, contacting the multi-spanning membraneprotein Sec6la (Figure 7). This finding suggests that thenascent chain is transferred directly from the ribosomeinto the translocation site (Figure 8). In the case of alate stage of translocation in which the signal sequencehas been cleaved off and in which the nascent chainhas adopted a trans-membrane orientation, the regioncontacting Sec6la was found to comprise -40 aminoacid residues. Within this region, Sec6la was the majorcrosslinking partner and, for two tested continuous poly-peptide segments, each residue could be crosslinked toSec6la. Since this membrane protein accounts for only asmall percentage of the total molecules in the ERmembrane and nevertheless gives most of the crosslinks,it seems to form a separated translocation site; othermembrane proteins cannot have easy lateral access to thenascent chain. The lack of other crosslinks is even moreremarkable if one takes into account that in our experi-mental set up polypeptides are trapped for a prolongedtime period during their transfer through the membrane.Our data would therefore suggest that Sec6la surroundsthe translocating polypeptide at least partially. The simplestexplanation for these findings would be a model in whichSec6la forms a protein-conducting channel. Such anassumption would fit with its multi-spanning structure andwould also explain best the electrophysiological data(Simon and Blobel, 1991). Also, a channel would beconsistent with the observation of a hydrophilic membraneenvironment of translocating preprolactin chains, asindicated by the short fluorescence life-time of incorpor-ated probes (Crowley et al., 1993). However, so farwe cannot exclude that part of the environment of atranslocating nascent chain is formed from lipids and/orthat Sec6la does not surround the nascent chain com-pletely. A related protein-conducting channel may exist

3978

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Fig. 7. Schematic summary of the results of the systematic crosslinking experiments. The scheme summarizes the results of the crosslinkingexperiments with the 187mer and 169mer from which the signal sequence is cleaved off (187mer-SP and 169mer-SP) and with the 132mer fromwhich the signal sequence is not cleaved. The results for the 86mer are not included because only a few positions were analyzed for crosslinking.The numbers below the lines give the positions of the nascent chains at which single photoreactive groups were introduced. The black bars indicateregions of the nascent chain which give crosslinks to Sec6la. The positions at which crosslinks to the TRAM protein and to proteins of 70, 30 and27 kDa are observed are also indicated. All nascent chains are aligned with their C-terminus located in the peptidyl transferase centre of theribosome.

in bacteria, since SecYp, the equivalent to Sec6la, is alsoadjacent to polypeptides trapped in translocation andshields them from phospholipids (Joly and Wickner, 1993).Our conclusion that Sec6la is the major membrane proteinadjacent to translocating chains is in agreement withthe fact that some proteins, like preprolactin, can betranslocated into proteoliposomes containing only the SRPreceptor (probably required for the targeting process only)and the Sec6lp complex (Gorlich and Rapoport, 1993).The segment of the nascent chain contacting Sec6 1a

was shifted in accordance with the length of the polypep-tide (Figure 7), suggesting that strong specific interactionsbetween certain regions of both partners do not exist.Nevertheless, since in some cases crosslinking fromneighboring positions differed greatly in intensity, itseems that there are preferred interactions or crosslinkingreactivities. Crosslinking to a small unidentified membraneprotein of 7-8 kDa showed a particular positional effect.A similar crosslink has been observed with other transloca-tion substrates (Kuroiwa et al., 1993). The crosslinkingpartner may be Sec6ly (-8 kDa) or RAMP 4, a ribosome-associated membrane protein of 10 kDa (Gorlich andRapoport, 1993) (immunoprecipitation experimentsexclude Sec6l j; data not shown).The length of the polypeptide chain that is in contact

with Sec6la (-40 residues for the 169mer; -40-50residues for a 153mer and a 178mer; data not shown) issurprisingly long since only 20 amino acid residues would

be expected to be located in the plane of the phospholipidbilayer if they adopted an a-helical structure, and even

fewer residues if they were in a more extended conforma-tion. Structural fluctuation of some portions of the transloc-ating polypeptide chain may be one explanation, but it isperhaps more likely that parts of the cytoplasmic and/or lumenal loops of the Sec61a molecule contact thetranslocating polypeptide and form extensions of theputative membrane channel (see scheme in Figure 8).Our conclusion, that during cotranslational translocation

the nascent chain is transferred directly from the ribosomeinto the membrane, is in agreement with evidence indicat-ing a tight and specific association of the Sec6lp complexwith membrane-bound ribosomes. The ribosome-Sec61pinteraction is induced by the membrane targeting of a

nascent chain and is probably disrupted by the dissociationof the ribosome into its two subunits (Gorlich et al.,1992b). The ribosome protects Sec6la from the attack byproteases (Kalies et al., 1994). Since fluorescent probesincorporated into translocating nascent chains cannot bequenched by iodide added to the cytosolic compartment(Crowley et al., 1993), it seems that a tight seal is producedbetween the ribosome and the membrane, preventing theleakage of small molecules. Although it is not yet clearwhether the Sec6lp complex is the only componentparticipating in the formation of the seal, it seems possiblethat the ribosome makes numerous contacts with Sec61a,perhaps with the cytosolic loops of the latter, thereby

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Fig. 8. A model for the cotranslational translocation process. Duringtranslocation, nascent preprolactin chains are transferred directly fromthe ribosome into the membrane. At early phases of the process, thepolypeptides are assumed to be inserted in a loop structure into achannel formed from Sec6la. The N-terminus of the signal sequencecontacts the TRAM protein (86mer and 132 mer). After signalsequence cleavage the nascent chain spans the membrane inside achannel formed from Sec6la (169mer and 187mer; the latter is notshown). Several other membrane proteins seem to contact thepolypeptide as it emerges at the lumenal side of the ER membrane.The approximate numbers of amino acid residues of the nascent chainslocated inside the ribosome and in contact with Sec6la and othermembrane proteins are indicated.

leading to an extension of the protein-conducting channelof the ribosome into the membrane.

With early translocation intermediates corresponding toshort nascent chains with uncleaved signal sequences, theregion of the polypeptide chain preceding the ribosome-buried portion was also found to contact Sec6la. However,for the longer of the investigated chains (l32mer) thesegment in proximity to Sec6la was found to be abouttwice as long as that for the 169mer (80 versus 40 residues;Figure 7). A possible explanation is that the nascent chainis inserted into the membrane in a loop structure in whichit spans the membrane twice inside an environment formedfrom Sec6la (Figure 8). A loop structure would beconsistent with the fact that the 132mer has a lengthonly slightly shorter than that required for signalsequence cleavage to occur (143 amino acids; S.Voigt and

T.A.Rapoport, unpublished results), so that the cleavagesite (position 31) may be expected to be close to thelumenal side of the membrane where the active site ofthe signal peptidase is located. The assumption of aloop structure is also supported by data that indicate acytoplasmic location of the N-terminal region of a secret-ory protein (Shaw et al., 1988).The region of the signal sequence preceding its hydro-

phobic core is mainly in contact with the TRAM protein,in agreement with previous results (High et al., 1993b)(Figure 7). A minor crosslink to TRAM was also observedfor the 132mer with the photoreactive probe in position31, whereas all other positions did not give crosslinks.Moreover, crosslinks to the TRAM protein were notobserved from any position of a late translocation inter-mediate. Thus, TRAM seems to sense the presence of asignal sequence and to interact specifically with regionsflanking its hydrophobic core. It may perhaps play a rolein orienting the signal sequence in a loop structure in themembrane. Since the N-terminus of both the 86mer andthe 132mer was found to interact with the TRAM protein,one may assume that it is fixed at the cytoplasmic side ofthe membrane, while the C-terminal portion of the loopmoves through the membrane as an elongating hairpin.After signal peptide cleavage, the nascent chain wouldadopt a transmembrane orientation and the TRAM proteinwould disengage (see Figure 8).An unexpected finding is the occurrence of crosslinks

from certain positions of the nascent chain to othermembrane proteins in the lumen of the ER. One minorcrosslinking partner with an apparent molecular mass of27 kDa could be identified as TRAPax (previously calledSSRa; Hartmann et al., 1993), in agreement with previousdata which demonstrated the crosslinking of translocatingchains to TRAPa (Wiedmann et al., 1989; Gorlich et al.,1990, 1992a). One major unidentified lumenal crosslinkingpartner is a glycosylated membrane protein of 70 kDawhich probably corresponds to a crosslinking partnernoted by Kellaris et al. (1991) and Thrift et al. (1991).The function of these membrane proteins is unclear atpresent. They may be involved in folding of the nascentpolypeptide chain, in modification reactions or in therelease of polypeptides from the translocation site.

Materials and methodsMaterialsSP6 polymerase, protein A-, protein G- and concanavalin A-Sepharose, as well as deoxynucleotide triphosphates, were purchasedfrom Pharmacia. Nickel nitrilo-tri-acetic acid (Ni NTA) agarose, puromy-cin and yeast tRNA were obtained from Qiagen, Sigma and BoehringerMannheim, respectively. [35S]methionine and [a-35S]ATP were obtainedfrom Amersham; Taq polymerase, RNAsin and the in vitro mutagenesiskit were purchased from Promega. Trifluoromethyl-diazarinobezoic acid(TDBA) was a kind gift from Dr J.Brunner.TDBA-lysyl tRNA was produced as described (Gorlich et al., 1991)

and purified by BD-cellulose chromatography.

MutagenesisThe plasmid pGEMBP1 was cut with the restriction enzymes BalI andSmaI and religated, resulting in the deletion of the 3' portion of thepreprolactin gene (Gorlich et al., 1992b). The shortened gene wasexcised with Hindu]I and EcoRI and cloned into the vector pAlter(Promega). Using appropriate oligonucleotides, the lysine codons atpositions 154, 136, 99 and 78, 72 were altered to asparagine and argininecodons, respectively. The resulting modified preprolactin gene was

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cloned into the plasmid pGEM2. In a separate mutagenesis step, thelysines at positions 4 and 9 of the signal sequence of the wild typepreprolactin gene were mutagenized to arginines. Subsequently, thealtered signal sequence was excised with BglI and PpuMI and ligatedto the mutagenized mature region, yielding a lysine null mutant. Aftercloning into pAlter, single AAA-lysine codons were introduced into thisgene at the following positions using appropriate oligonucleotides: 151,147, 143, 139, 136, 135, 134, 133, 132, 131, 127, 123, 118, 117, 116,115, 114, 112, 111, 107, 103, 99, 95, 91, 87, 83, 78, 75, 72, 66, 63,61, 59, 55, 47, 31 and 18 (numbering from the N-terminus of thesignal sequence).

TranscriptionAll mutants in the plasmid pAlter were directly transcribed with SP6RNA polymerase. Transcripts coding for the first 86, 132 or 169 aminoacids of preprolactin (86mer, 132mer or 169mer) were produced aftercutting the plasmids with PvuII, RsaI or EcoRI, respectively. Transcriptscoding for 187 amino acids (187mer) were produced from a templateobtained by PCR, using as the 5' primer an oligonucleotide correspondingto the SP6 promoter and as the 3' primer an oligonucleotide correspondingto the T7 promoter region of the plasmid. The latter was altered toremove two stop codons and to extend it by six codons for histidines.The resulting amino acid sequence at the C-terminus from position 164to 187 reads: FGGTELEFALYRVVLGSHHHHHHG. After the PCR, theDNA was purified by agarose electrophoresis and transcribed with SP6RNA polymerase.

Translation and photocrosslinkingTranslation of the transcripts in the wheat germ system was carried outessentially as described (Gorlich et al., 1992b). A translation mixture of10 gl, containing 50 nM SRP, 10 giCi [35S]methionine and two equivalents[for a definition see Walter et al. (1981)] puromycin/high salt-strippedrough microsomes (PK-RM), was incubated at 26.5°C for 2 min; TDBA-lysyl tRNA (1.5 pmol) was added and the reaction was started by theaddition of transcripts. After 2 min, 4 ItM edeine was added and theincubation was continued for different periods of time, depending onthe length of the polypeptide fragment (5 min for the 86mer, 10 min forthe 132mer, 14 min for the 169mer and 16 min for the 187mer).Irradiation was carried out for 10 min with a long wavelength UV lamp(Blak-Ray, model B1000 A). Immediately after irradiation, the sampleswere incubated with I or 2 mM puromycin at 500 mM potassium acetateand 2 mM magnesium acetate, first for 10 min on ice followed by10 min at 26.5°C. This procedure, followed by alkali extraction, removedall of the peptidyl-tRNA which otherwise was seen as two labeled bandsin SDS gels (see Figure 4, where the removal was incomplete owing tothe lack of alkali extraction).

Product analysisExtraction of the membranes with carbonate was carried out with0.5 ml 0.1 M sodium carbonate, adjusted to pH 12.5. The samples werecentrifuged for 11 min at 75 000 r.p.m. in a table top ultracentrifuge(Beckman) using the 100.3 rotor. The pellets were dissolved in SDSsample buffer.The binding of crosslinked products to concanavalin A-Sepharose

was carried out as follows. To 10 gl of a translation mixture, 20 pl of2% SDS in 50 mM Tris-HCl pH 8.0 were added. The mixture wasincubated for 15 min at 55°C, diluted with 0.4 ml of concanavalin Abuffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 1 mM each of MgCl2,MnCl2, CaCl2 2% Tween 20) and incubated with 20 1l concanavalinA-Sepharose overnight at 4°C. The resin was washed twice withconcanavalin A buffer, once with water and the bound material waseluted with SDS sample buffer. The efficiency of binding was 40% forthe crosslinked product containing the 70 kDa protein and 15-20% forthat containing TRAPa.

For immunoprecipitation of crosslinked products, samples of 10 pltranslation mixture were denatured with SDS as described before anddiluted with 0.4 ml I buffer (50 mM Tris-HCI pH 7.5, 0.5 M NaCl,2% Triton X-100). Then 20 gl of antibodies, covalently linked to a 6:1mixture of protein A- and protein G-Sepharose, were added whichwere preincubated with 20% bovine serum albumin for 20 min. Afterincubation overnight at 4°C the resin was washed twice with I bufferand once with water, and the bound material was eluted with SDS-sample buffer. Immunoprecipitation of Sec6la was carried out with amixture of antibodies directed against the N- and C-termini of the protein(Gorlich et al., 1992b). The efficiency of immunoprecipitation was -40%,both for crosslinked and non-crosslinked Sec6la. Immunoprecipitation of

TRAM was carried out with antibodies against its C-terminus (Gorlichet al., 1992a). The efficiency of immunoprecipitation was -60%.

Binding of the crosslinked products of the 187mer to Ni NTA agarosewas carried out as follows. Samples of 10 gil translation mixture werediluted with 0.4 ml B buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris,adjusted to pH 8.0 with NaOH) containing 1% Triton X-100, and 15 g1Ni NTA agarose were added. After agitation for 30 min at roomtemperature, the resin was washed twice with C buffer (same as B bufferbut adjusted with HCI to pH 6.3) containing 1% Triton and once withC buffer without detergent. Elution of the bound material was carriedout with 20 ml C buffer containing 0.1 M EDTA for 5 min at roomtemperature followed by an equal volume of a double concentratedSDS-sample buffer.

Proteins were separated in 10-20% linear acrylamide gels and visual-ized by fluorography or by analysis with a Fuji PhosphorimagerBAS2000. For each experiment, the amount of radioactivity in non-crosslinked nascent chains was determined by phosphorimaging andequal counts were loaded on each lane of a second gel. Exposure timesof the X-ray films were generally between 3 and 10 days, except for theexperiments in Figures 3B and 4A (21 days). The exposure time for thephosphorimage shown in Figure 4B was 7 days.

AcknowledaementsWe gratefully acknowledge the technical help of E.Burger and B.Nentwig.We thank K.Jurchott for the construction of a plasmid, D.Gorlich,L.Dreier and A.Wittstruck for providing coupled antibodies, andE.Hartmann, B.Jungnickel, B.Dobberstein, S.High and S.M.Rapoport forcritical reading of the manuscript. We are particularly grateful toJ.Brunner for the gift of TDBA. This work was supported by grantsfrom the DFG, the BMFT, the Fonds der Chemischen Industrie and theHuman Science Frontier Program.

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Received on April 25, 1994; revised on June 14, 1994

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