Discrete Cross-linking Products Identified during Membrane Protein Biosynthesis*

(Received for publication, May 29, 1996, and in revised form, October 4, 1996)

Vivienne Laird Dagger and Stephen High §

From the School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

We have investigated the molecular details of the membrane insertion of the multiple-spanning membrane protein opsin. Using heterobifunctional cross-linking reagents the endoplasmic reticulum (ER) proteins adjacent to a series of defined translocation intermediates were determined. Once the nascent opsin chain reaches a critical minimum length Sec61alpha is the major ER component adjacent to the polypeptide. Using a homobifunctional reagent, the cross-linking partners from a single cysteine residue in the nascent chain were analyzed. This approach identified chain length-dependent cross-linking products between nascent opsin and a 21-kDa ribosomal protein, followed by Sec61beta and finally with Sec61alpha . Our data support a model where the sequential transmembrane domains of a multiple-spanning membrane protein are integrated at an ER insertion site similar to that mediating the insertion of single-spanning membrane proteins.


INTRODUCTION

Membrane proteins are targeted to the endoplasmic reticulum (ER)1 membrane by a signal sequence which is usually a stretch of up to 20 apolar amino acid residues (1). The targeting process involves the recognition and binding of these signals by the signal recognition particle (SRP) (2). At the membrane, SRP interacts with the SRP receptor and, in a GTP-dependent manner, presents the nascent chain to the translocation/insertion machinery (2, 3).

Cross-linking studies have been used to identify ER proteins responsible for the translocation of secretory proteins and the insertion of single-spanning membrane proteins (reviewed in Ref. 4). Using a photocross-linking approach, Sec61alpha was identified as a major cross-linking partner of the secretory protein preprolactin (5-7) and type I and type II membrane proteins (8-11). Sec61alpha was also identified as a major cross-linking partner of secretory and membrane proteins using bifunctional cross-linking reagents (10, 12, 13). Sec61alpha is part of a protein complex, together with Sec61beta and Sec61gamma (14, 15), and reconstitution studies showed that this Sec61 complex plus the SRP receptor are essential components for secretory protein translocation and membrane protein insertion (14, 16). Cross-linking also identified a second component of the translocation machinery, the translocating chain-associating membrane protein (TRAM) (8, 9, 11, 17-19). Reconstitution studies showed that TRAM is required for the efficient insertion/translocation of a subset of membrane and secretory proteins (14, 16, 19). While initial studies allowed cross-linking from a number of positions within the nascent chain, more recently site-specific cross-linking techniques have been developed, allowing the environment of particular regions of the nascent chain to be investigated (6-8, 20, 21).

Previous cross-linking studies have concentrated on secretory proteins, and simple, single-spanning membrane proteins. In this study we have analyzed the biosynthesis of opsin, a 39-kDa multiple-spanning membrane protein which is targeted to the ER membrane by SRP (22, 23). Truncated mRNAs were used to generate "translocation intermediates" which remain associated with the ER translocation site due to the presence of the ribosome at the C terminus of the truncated polypeptide (24). Hetero- and homobifunctional cross-linking reagents were then used to identify cross-linking partners of the inserting nascent chain. With heterobifunctional reagents the only component of the ER translocation site cross-linked to nascent opsin chains was Sec61alpha . This adduct was only observed when the nascent chain reached a critical minimum length. In contrast, using the homobifunctional reagents we observed discrete chain length-dependent cross-linking products between the nascent chain and a 21-kDa ribosomal protein, followed by Sec61beta , and finally Sec61alpha .


EXPERIMENTAL PROCEDURES

Materials

The plasmid coding for opsin, pGEM3OP, was kindly provided by Reid Gilmore, University of Massachusetts, Worcester, MA. BsaHI, used to generate the 155-amino acid truncation of opsin and the Endoglycosidase H (recombinant fusion protein) (Endo Hf) were from New England Biolabs (Hitchin, Herts). [35S]Methionine was supplied by Amersham (Amersham, Bucks) and T7 RNA polymerase by Promega (Southampton, Hants). All cross-linking reagents were purchased from Pierce & Warriner (Chester, Cheshire) and Apollo Chemicals Ltd. (Stockport, Cheshire). The concanavalin A-Sepharose was purchased from Pharmacia Biotech (St. Albans, Herts). All other chemicals were purchased from BDH/Merck (Poole, Dorset) and Sigma (Poole, Dorset). The antibodies specific for SRP54, Sec61beta , TRAM, and Sec61alpha (under denaturing conditions), were a gift from Bernhard Dobberstein, ZMBH, Heidelberg, Germany. The Sec61alpha antisera used under "native" conditions, and the Sec61gamma antisera, were both raised by Research Genetics Inc. (Huntsville, AL) using peptides encoding the C-terminal (alpha ) and N-terminal (gamma ) 12 amino acids of the published sequences.

Construct Preparation

The coding region of bovine opsin was subcloned from pGEM3OP into the plasmid pGEM3z using an EcoRI/HindIII fragment. Most of the templates for the transcription of different truncated opsin mRNAs were prepared by PCR (25). The resulting opsin translocation intermediates were all efficiently membrane integrated, glycosylated, and immunoprecipitated by an anti-opsin monoclonal antibody. The upstream primer was 160 bases 5' of the T7 RNA polymerase promoter and had the sequence 5'-GGGCCTCTTCGCTATTACGC-3', antisense primers were designed to make 5 truncations of 106, 127, 132, 137, and 150 amino acids. For the 106-amino acid truncation (OP106) the primer 5'-GGCCCAAAGACGAAGTACCC-3' was used, 127-amino acid truncation (OP127) 5'-GGACCACAGTGCAATTTCAC-3', 132-amino acid truncation (OP132) 5'-GGCCAGGACCACCAAGGACCA-3', 137-amino acid truncation (OP137) 5'-ACCACGTACCGCTCGATGGC-3', and for the 150-amino acid truncation (OP150) the primer 5'-CTCCCCGAAGCGGAAGTTGCT-3' was used. The PCR products were purified directly from the reaction mixture using the Wizard PCR purification kit (Promega, Southampton, Hants.). The template for the 155-amino acid truncation (OP155) was made by cleavage of the plasmid within the coding region using the restriction endonuclease BsaHI.

Site-directed Mutagenesis

The cysteine residue at position 140 was altered to a glycine using the Clontech Laboratories site-directed mutagenesis kit (Palo Alto, CA). Subsequent sequencing revealed two discrepancies from the published sequence (26), these were single base changes in the codons for amino acids 101 and 118 and did not alter the amino acid residue encoded. These changes were also present in the original plasmid.

In Vitro Transcription and Translation

Transcription of the purified DNA was carried out as described by the manufacturer (Promega, Southampton, Hants.). Translation of the resulting transcripts in a rabbit reticulocyte lysate system (Promega, Southampton, Hants.) was carried out at 30 °C in the presence of [35S]methionine and canine pancreatic microsomes as described by the manufacturer. Translation initiation was inhibited after 15 min by the addition of 4 mM 7-methylguanosine 5'-monophosphate, and chain elongation allowed to continue for a further 10 min until translation was inhibited by the addition of 2 mM cycloheximide.

Cross-linking with Bifunctional Reagents, Immunoprecipitation, and Sample Analysis

For cross-linking with bifunctional reagents, the membrane associated components were isolated by centrifugation through a high salt/sucrose cushion (250 mM sucrose, 500 mM KOAc, 5 mM Mg(OAc)2, and 50 mM HEPES-KOH, pH 7.9) for 10 min at 4 °C and 55,000 rpm (approx 100,000 × g in a TLA100.2 rotor, Beckman instruments). The resulting pellet was resuspended in a low salt/sucrose buffer (250 mM sucrose, 100 mM KOAc, 5 mM Mg(OAc)2, and 50 mM HEPES-KOH, pH 7.9), a sample taken for trichloroacetic acid precipitation, then N-hydroxysuccinimidyl iodoacetate, succinimidyl 4-(p-maleimidophenyl)butyrate, MBS, BMP, or BMH added to a final concentration of 1 mM, or S-MBS added at 0.12 mM (27). Following incubation for 10 min at 26 °C, samples were quenched with 100 mM glycine, 5 mM 2-mercaptoethanol or 5 mM 2-mercaptoethanol alone (BMP and BMH). A second sample was removed for trichloroacetic acid precipitation. In order to establish whether PCR-induced mutations had any effect; mRNAs derived from independent PCR reactions were translated and the cross-linking products were compared. No differences between independent experiments could be detected.

Denaturing immunoprecipitations were performed by heating the samples for 5 min at 95 °C in the presence of 1% SDS. Native immunoprecipitations were performed by releasing the ribosome from the nascent chain by incubation with 1 mM puromycin and 400 mM KCl for 10 min at 30 °C. Four volumes of immunoprecipitation buffer (10 mM Tris/HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) were then added to all samples and aliquots were incubated overnight at 4 °C with the relevant antisera in the presence of 0.2 mg/ml phenylmethylsulfonyl fluoride and 1 mM methionine. Protein A-Sepharose was added for 2 h and samples processed as described previously (see Ref. 28). All samples were analyzed on 12% SDS-polyacrylamide gels and exposed overnight to a PhosphorImaging plate for visualization on a Fuji BAS-2000 PhosphorImaging system. Concanavalin A binding was performed as described by Krieg et al. (18).


RESULTS

Topology of the Nascent Opsin Translocation Intermediates

In this study opsin "translocation intermediates" were used to investigate the insertion of a model multiple-spanning protein into the ER membrane using cross-linking techniques. A translocation intermediate is a truncated nascent chain which, due to the lack of a stop codon in the truncated mRNA, is not released from the ribosome and becomes trapped in the translocation machinery (24).

Prior to cross-linking analysis, the topology of the 155-amino acid long opsin truncation (OP155cko) was established using a protease protection assay (Fig. 1a). Nascent chains protected against protease digestion were detected by immunoprecipitation with an antibody specific for the N terminus of opsin. Proteinase K digestion of OP155 after puromycin release resulted in a large protected fragment which migrated slightly faster than the undigested form (OP155-2CHO) (Fig. 1a, cf. lane 2, asterisk, with lane 1). This altered mobility is due to cleavage of the exposed C terminus (cf. Fig. 1b). Upon Endo Hf digestion, this large protected band shifted to migrate with a mobility similar to the nonglycosylated nascent chain (OP155) (Fig. 1a, lane 3, asterisk). A fraction of the proteinase K-treated material had a smaller molecular weight after Endo Hf treatment (Fig. 1a, lane 3) indicating that there was some cleavage within the cytosolic loop of OP155 (cf. Fig. 1b). Digestion with trypsin after release of the ribosome also resulted in a large protected fragment (Fig. 1a, lane 5, asterisk). In this case no cleavage of the C terminus was apparent and cleavage in the cytosolic loop was again relatively inefficient, despite two potential cleavage sites for trypsin at lysine residues 66 and 67 (cf. Fig. 1b). However, trypsin digestion of the intact translocation intermediate (i.e. in the presence of the ribosome) significantly enhanced cleavage within the cytosolic loop to produce the fragment OP67-2CHO (Fig. 1a, lane 6, asterisk). Endo Hf digestion resulted in a ~7-kDa fragment corresponding to an N-terminal polypeptide of ~67 residues with both carbohydrate side chains removed (OP67) (Fig. 1a, lane 7, asterisk). Thus, the first transmembrane domain of the opsin translocation intermediate is fully integrated in the correct orientation with the N terminus glycosylated and the cytosolic loop region sensitive to protease. Nonglycosylated OP155 chains (see Fig. 1a, lane 1) are not protease resistant and are therefore not membrane integrated but are most likely ribosome-bound material which co-isolates with the membrane fraction (see later). The predicted cytosolic loop between the first and second transmembrane domains is particularly sensitive to trypsin when the nascent chain is still ribosome bound suggesting that after release the polypeptide chain assumes a different conformation (cf. Fig. 1b). Control digestions in the presence of Triton X-100 resulted in total loss of detectable nascent chain (Fig. 1a, lanes 4 and 8). Similar analysis showed the 127-amino acid nascent chain (OP127) is also correctly inserted with the N terminus glycosylated and protease protected (data not shown).


Fig. 1. a, protease protection of OP155 translocation intermediates. OP155 translocation intermediates were incubated with proteinase K at 500 µg/ml (lanes 1-4) or trypsin at 250 µg/ml (lanes 5-8) for 30 min on ice and protected fragments immunoprecipitated with antisera specific for the N terminus of opsin. After immunoprecipitation, some samples (lanes 3 and 7) were treated with Endo Hf. b, proposed topology of the OP155 translocation intermediate. The OP155 translocation intermediate appears to have a fully integrated first transmembrane domain (zigzag line), with the second and third transmembrane domains synthesized, but only partially inserted. Dissociation of the ribosome leads to integration of the second and third transmembrane regions. c, putative translocation intermediates of different length opsin nascent chains. The estimated positions of the lysine residues (K) and cysteine residue 110 (black dot) are indicated. The position of the cysteine and lysine residues were calculated assuming that 35-40 amino acid residues are buried in the ribosome (see Ref. 37) and 20 amino acids span the membrane (50 Å) (see Ref. 18). Bovine opsin normally has two cysteine residues in the first 155 amino acids of the polypeptide chain, at positions 110 and 140. The cysteine residue at position 140 was altered to a glycine residue leaving only a single cysteine remaining at position 110. This is denoted by the suffix "cko."
[View Larger Version of this Image (26K GIF file)]


Sec61alpha Is Adjacent to Multiple-spanning Proteins during Membrane Insertion

To investigate the proteins adjacent to membrane inserting opsin nascent chains the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (reacting principally with cysteine and lysine residues, see Ref. 13) was added to opsin translocation intermediates. The two shortest chains analyzed were 106 and 137 amino acids in length (OP106 and OP137, respectively), these translocation intermediates have pronounced peptidyl-tRNA species of ~32 and ~37 kDa as previously reported for other nascent chains (7, 29). These peptidyl-tRNA species are lost upon puromycin treatment of the samples (compare Fig. 2a, lanes 9 and 10 with Fig. 5, lanes 1 and 2). Both OP106 and OP137 were only found to be cross-linked to SRP54 (Fig. 2a, lane 4 and 12, open arrow). Only when the nascent chain was 155 amino acids in length (OP155cko) was cross-linking to Sec61alpha observed (Fig. 2a, lane 21, closed arrow). Sec61alpha was visible as a doublet within the molecular mass range 46-60 kDa (Fig. 2a, lanes 18, and 21, closed arrow) similar to cross-linking products previously identified using bifunctional reagents (see Refs. 10 and 12). This doublet probably reflects cross-linking of the nascent chain to different sites within Sec61alpha .2 No cross-linking of Sec61beta alone was visible (cf. Fig. 3), however, a high molecular weight product was specifically immunoprecipitated with anti-Sec61beta serum under denaturing conditions (Fig. 2a, lane 22, unfilled arrow). This product must therefore include the nascent chain, Sec61beta and at least one unidentified protein. A high molecular weight product was also immunoprecipitated with antisera recognizing TRAM (Fig. 2a, lane 23, broad band), again suggesting multiple cross-linking.


Fig. 2. a, cross-linking of different length nascent opsin chains to ER components using MBS. Opsin translocation intermediates were incubated in the presence of 1 mM MBS (+) or mock treated with dimethyl sulfoxide (-). After quenching with 100 mM glycine and 5 mM 2-mercaptoethanol, total products were trichloroacetic acid precipitated (lanes 1, 2, 9, 10, 17, and 18). The rest of the samples were solubilized in immunoprecipitation buffer and incubated overnight with antisera against known components of the ER targeting and translocation machinery. Cross-linking products to SRP54 (open arrow), to Sec61alpha (closed arrow), and a high molecular weight complex incorporating Sec61beta (unfilled arrow) are indicated. A dot denotes the diglycosylated opsin nascent chain. b, cross-linking with S-MBS. Translocation intermediates were incubated with 0.12 mM S-MBS (+) (27) or mock treated with water (-). After quenching, total products were trichloroacetic acid precipitated (lanes 1, 2, 9, 10, 17, and 18) or immunoprecipitated as above.
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Fig. 5. The Sec61beta cross-linking product is from stably inserted nascent chains. OP137 translocation intermediates were cross-linked to Sec61beta using BMH and products immunoprecipitated with antisera specific for opsin (lanes 1 and 2) or Sec61beta (lanes 3 and 4). Samples in lanes 1-3 were then analyzed directly. The products immunoprecipitated in lane 4 were eluted from the protein A-Sepharose and further analyzed for concanavalin A-Sepharose binding prior to electrophoresis. For symbols, see previous figure legends.
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Fig. 3. Cross-linking with the cysteine specific reagent BMP. Opsin translocation intermediates were incubated with 1 mM BMP (+) or mock treated with dimethyl sulfoxide (-). After quenching with 5 mM 2-mercaptoethanol, total products were trichloroacetic acid precipitated (lanes 1, 2, 9, 10, 17, 18, 25, 26, 33, 34, 41, and 42) or immunoprecipitated as indicated. An asterisk indicates a novel 21-kDa cross-linking partner. For other symbols, see legend to Fig. 2a.
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Bifunctional reagents similar to MBS but with shorter (N-hydroxysuccinimdyl iodoacetate) and longer (succinimidyl 4-(p-maleimidophenyl)butyrate) spacer arms were also used. Both gave very similar results to MBS showing that the length of the spacer arm did not affect the cross-linking partners detected (data not shown). However, when S-MBS, a water soluble analogue of MBS was used, fewer cross-linking products were visible (Fig. 2b). SRP54 was still a cross-linking partner of all three nascent chains (Fig. 2b, lanes 4, 12, and 20, open arrows), but with OP155cko, only Sec61alpha was detected (Fig. 2b, lanes 18 and 21, closed arrow). None of the higher molecular weight species immunoprecipitated by antisera against Sec61beta and TRAM were visible (Fig. 2b, lanes 22 and 23) suggesting that penetration of the bilayer by the cross-linking reagent was necessary to obtain these products, but not for cross-linking to Sec61alpha .

Our principal conclusion from this initial analysis is that Sec61alpha is only seen as a major cross-linking partner when the opsin nascent chain is 155 amino acids in length. This result was unaffected by either the length of the spacer arm, or the solubility of the reagent used. The simplest interpretation of these results is that cross-linking of nascent opsin to ER membrane components occurs primarily from cysteine residue 110 of the nascent chain, and that this residue is only close to Sec61alpha when it has entered the plane of the membrane (cf. Fig. 1c).

Nascent Opsin Shows Discrete Cross-linking Products during Membrane Insertion

To further investigate ER proteins adjacent to cysteine residue 110, two cysteine specific cross-linking reagents with different length spacer arms, bismaleimidopropane (BMP, 11 Å) and bismaleimidohexane (BMH, 16.1 Å), were used. All known components of the ER translocation site have one or more cysteine residues (5, 15, 17), therefore the use of cysteine-specific homobifunctional reagents does not in principle restrict the cross-linking partner.

When opsin translocation intermediates were isolated and incubated with the cross-linking reagent BMP, specific cross-linking products were observed dependent upon the length of the translocation intermediate (Fig. 3). OP106 contained no cysteine residue, and no cross-linking products were observed (Fig. 3, lanes 1-8). OP127, OP132, and OP137 all formed cross-linking products of apparent molecular mass ~42 kDa. Subtracting the contribution of the nascent chain, this indicated each opsin translocation intermediate was cross-linked to a protein of ~21 kDa (Fig. 3, lanes 10, 18, and 26, asterisk) which was not a known component of the ER targeting or translocation machinery. In contrast, OP137, OP150cko, and OP155cko all showed discrete cross-linking products with Sec61beta (Fig. 3, lanes 30, 38, and 46, diamond). The first obvious evidence of Sec61alpha cross-linking products was only obtained when OP155cko was used (Fig. 3, lane 45, closed arrow). The Sec61alpha cross-linking product was a broad doublet similar to that seen with MBS and S-MBS. In addition to the nascent chain-Sec61beta adduct, a high molecular weight product identical to that observed with MBS was also seen (Fig. 3, lane 46, unfilled arrow, cf. Fig. 2a, lane 22). Analysis using BMH gave similar results, again showing that the cross-linking partners were not limited by the spacer arm length of the cross-linking reagent used (data not shown).

A number of opsin translocation intermediates are cross-linked to SRP54 (Figs. 2, a and b, and 3). Using a floatation assay (30) to separate membrane targeted nascent chains from non-targeted chains, we analyzed the OP155-SRP54 adducts and found these products fractionated with non-targeted nascent chains. In contrast, the Sec61alpha and Sec61beta cross-linking products were present in the membrane fraction (data not shown). We conclude that the SRP54 adducts arise from ribosome bound opsin chains which are pelleted with the membrane fraction prior to cross-linking, but which have not interacted productively with the ER microsomes used (see also results of protease protection above). Such incomplete release of SRP54 by canine pancreatic microsomes has been previously reported (10).

The reticulocyte lysate translation system has been reported to generate cross-linker-independent adducts under some circumstances (31). When control immunoprecipitations were performed without adding cross-linking reagents, a weakly labeled OP155-SRP54 adduct was observed (data not shown). No such products were immunoprecipitated with any of the other antisera used in this study confirming that the 21-kDa protein, Sec61beta and Sec61alpha are all bona fide cross-linking partners.

The 21-kDa Cross-linking Partner of Short Opsin Translocation Intermediates Is a Ribosomal Protein

Cross-linking of OP127, OP132, and OP137 with BMP resulted in a single major cross-linking partner of 21 kDa (Fig. 3, lanes 10, 18, and 26, asterisk) which was not a known component of the ER insertion machinery (see Fig. 3, lanes 11-16, 19-24, and 27-32) but was immunoprecipitated with a monoclonal antibody specific for opsin (data not shown). Cysteine residue 110 of OP127 is predicted to be deeply buried in the ribosome (Fig. 1c) and if the first transmembrane domain of OP127 is correctly inserted into the membrane the nascent chain should be glycosylated and hence any cross-linking products sensitive to Endo Hf digestion (Fig. 4). Endo Hf digestion does indeed increase the mobility of both the OP127 cross-linking product (Fig. 4, lane 4, asterisk) and the diglycosylated nascent chain (indicated with a dot). Thus, a correctly inserted and glycosylated OP127 translocation intermediate is cross-linked to a 21-kDa protein. The 21-kDa component was still a cross-linking partner in the absence of membranes (Fig. 4, lane 6), indicating that the 21-kDa protein is of ribosomal origin. The cross-linking product was not immunoprecipitated by antibodies recognizing the beta  subunit of the nascent polypeptide-associated complex, a previously identified 21-kDa ribosome-associated protein (30, 32).


Fig. 4. Characterization of the 21-kDa cross-linking partner of OP127. OP127 was synthesized in the presence (lanes 1-4) or absence (lanes 5 and 6) of microsomal membranes. After cross-linking with BMH, samples were analyzed directly after trichloroacetic acid precipitation (lanes 1 and 2) or after Endo Hf treatment (lanes 3 and 4). The material loaded in lanes 3 and 4 is equivalent to three times that loaded in lanes 1 and 2. OP127 translated without membranes was incubated in the absence (lane 5) or presence (lane 6) of 1 mM BMH for 10 min. After quenching the ribosome/nascent chain complexes were isolated and analyzed directly. OP denotes the unglycosylated opsin nascent chain. For other symbols see previous figure legends.
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The Sec61beta Cross-linking Product Is from a Stably Inserted Opsin Translocation Intermediate

Cross-linking of OP137 with homobifunctional cysteine-specific reagents resulted in a discrete Sec61beta -OP137 cross-linking product (Fig. 3, lane 30, and data not shown). This product could be immunoprecipitated with antisera against both opsin (Fig. 5, lane 2, diamond) and Sec61beta (Fig. 5, lane 3). The cross-linking product bound to concanavalin A-Sepharose (Fig. 5, lane 4) and was specifically eluted by 0.5 M alpha -methyl-D-mannoside (data not shown) indicating the presence of a carbohydrate moiety. Since canine Sec61beta does not contain any potential N-linked glycosylation sites (15), the carbohydrate present on the cross-linking product is from the opsin nascent chain. Sec61beta is therefore cross-linked to a correctly inserted OP137 translocation intermediate. The OP137-Sec61beta cross-linking product was also found to be sensitive to Endo Hf digestion (data not shown).


DISCUSSION

The truncated opsin nascent chains used in this study were shown to insert into canine pancreatic microsomes and form bona fide translocation intermediates. Once the nascent chain was trapped in the translocation machinery it could be cross-linked to adjacent proteins.

Using heterobifunctional reagents, and nascent polypeptides ranging from 106 to 155 amino acid residues in length, SRP54 adducts were observed with all chain lengths. These products represent SRP-bound nascent chains which have failed to target to the membrane. In striking contrast, strong cross-linking of membrane translocation intermediates to Sec61alpha was only observed with OP155cko, the longest of the nascent chains studied. This result was independent of the spacer arm length and solubility of the heterobifunctional reagents used suggesting that the majority of cross-linking products obtained are from the -SH group of cysteine residue 110 in opsin to -NH2 groups in the adjacent ER protein (see Fig. 1c). We conclude that cysteine 110 is only sufficiently close to Sec61alpha for cross-linking when it is 45 amino acid residues from the peptidyl transferase site of the ribosome, i.e. within the plane of the membrane. This is consistent with Sec61alpha being the major protein constituent of the ER insertion and translocation site (4-7, 10, 12-14, 16).

A comparison of membrane permeable and membrane impermeable (water soluble) cross-linking reagents revealed a clear difference. Membrane permeable reagents generated discrete high molecular weight products specifically immunoprecipitated with antisera against Sec61beta and TRAM. These "multiple" cross-linking products must represent OP155cko-Sec61beta and OP155cko-TRAM products that are in turn cross-linked to other components that remain to be identified (see below). Such products were not seen when S-MBS was used although cross-linking to Sec61alpha was maintained. Hence, penetration of the bilayer is necessary to obtain the high molecular weight Sec61beta and TRAM products. Thus, analysis with heterobifunctional reagents fully supports the view that Sec61alpha is the major protein component of an aqueous channel (7, 33, 34) through which the nascent opsin chain is inserted into the ER membrane. Sec61beta and TRAM are adjacent to both the nascent chain and additional, as yet unidentified, proteins.

To investigate the environment of a discrete region of the nascent chain, the cross-linking partners of a single cysteine residue were identified at different stages of the translocation process (Fig. 1c) using BMP and BMH. The first detectable cross-linking partner of the opsin nascent chain was a 21-kDa putative ribosomal protein. The protein is not the beta  subunit of the nascent polypeptide-associated complex, and may be identical to a previously described ribosomal component detected as a photocross-linking partner of short nascent luciferase chains (32).

When the chain length of the translocation intermediate was increased to 137 amino acids, cross-linking to Sec61beta was observed. Sec61beta is part of the Sec61 complex composed of Sec61alpha , Sec61beta , and Sec61gamma (14) and is probably a tail-anchored membrane protein with a single cysteine present in the cytoplasmic N terminus (15). This is the first direct evidence that a membrane inserting nascent chain is adjacent to Sec61beta . Cross-linking to Sec61beta was shown to be from glycosylated, membrane inserted, translocation intermediates. Since the single cysteine residue present in OP137 is probably still within the ribosome (Fig. 1c) this suggests that the cytoplasmic domain of Sec61beta extends into the channel of the large ribosomal subunit.

After initial cross-linking of the nascent chain to Sec61beta alone, increasing the nascent chain length to 155 residues (OP155cko) allows cross-linking to both Sec61alpha and Sec61beta . Sec61alpha is proposed to be the major protein component of the ER translocation site (5-7, 10, 12, 13). Thus, these data support the view that the integration of multiple-spanning membrane proteins occurs at a general ER translocation site, very similar to that which promotes the insertion of single-spanning membrane proteins and the translocation of secretory proteins (4, 35).

In addition to adducts between OP155cko and Sec61beta alone, more complex high molecular weight products were observed with all cross-linking reagents used. These cross-linking products are not immunoprecipitated with antisera specific for Sec61alpha and therefore contain additional unidentified protein(s) which may be ribosomal (see above).

These results are consistent with the ribosome and the ER translocation site forming a continuous channel (33) where some ER protein components extend into the ribosomal channel (7, 36). Sec61alpha appears to be the major constituent of this membrane channel, our data suggest that Sec61beta may also contribute to the environment. Alternatively, Sec61beta may play a distinct role such as regulating the lateral exit of transmembrane domains from the ER translocation site into the lipid bilayer (4). Recent evidence has shown this to be a complex process which may involve TRAM (8).

The approach used in this study only enables the detection of proteins adjacent to the inserting opsin nascent chains. Recent photocross-linking analysis suggests that the translocation site is partly composed of phospholipid, perhaps promoting the lateral exit of hydrophobic transmembrane domains into the bilayer (20). Our results suggest that both Sec61alpha and Sec61beta are significant components of the translocation channel, however, they do not preclude the possibility that lipid also makes a significant contribution to the site of opsin integration at the ER membrane. We are presently attempting to address this question.


FOOTNOTES

*   This work was supported by funding from the Biotechnology and Biological Sciences Research Council, the Human Frontier Science Program Organisation, and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Biotechnology and Biological Sciences Research Council Research Studentship.
§   Biotechnology and Biological Sciences Research Council Advanced Research Fellow. To whom correspondence should be addressed. Tel.: 0161-275-5070; Fax: 0161-275-5082.
1    The abbreviations used are: ER, endoplasmic reticulum; BMH, bismaleimidohexane; BMP, bismaleimidopropane; cko, cysteine knock out; Endo Hf, Endoglycosidase H, recombinant fusion protein; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; OP, opsin; S-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; SRP, signal recognition particle; TRAM, translocating chain-associating membrane protein; PCR, polymerase chain reaction.
2    S. High, unpublished data.

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