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Signal recognition particle mediates post-translational targeting in eukaryotes

2004; Springer Nature; Volume: 23; Issue: 14 Linguagem: Inglês

10.1038/sj.emboj.7600281

1460-2075

Ben M. Abell, Martin Pool, Oliver Schlenker, Irmgard Sinning, Stephen High,

Endoplasmic Reticulum Stress and Disease

Article1 July 2004free access Signal recognition particle mediates post-translational targeting in eukaryotes Benjamin M Abell Benjamin M Abell School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Martin R Pool Martin R Pool School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Oliver Schlenker Oliver Schlenker Biochemiezentrum der Universität Heidelberg (BZH), Heidelberg, Germany Search for more papers by this author Irmgard Sinning Irmgard Sinning Biochemiezentrum der Universität Heidelberg (BZH), Heidelberg, Germany Search for more papers by this author Stephen High Corresponding Author Stephen High School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Benjamin M Abell Benjamin M Abell School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Martin R Pool Martin R Pool School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Oliver Schlenker Oliver Schlenker Biochemiezentrum der Universität Heidelberg (BZH), Heidelberg, Germany Search for more papers by this author Irmgard Sinning Irmgard Sinning Biochemiezentrum der Universität Heidelberg (BZH), Heidelberg, Germany Search for more papers by this author Stephen High Corresponding Author Stephen High School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Author Information Benjamin M Abell1, Martin R Pool1, Oliver Schlenker2, Irmgard Sinning2 and Stephen High 1 1School of Biological Sciences, University of Manchester, Manchester, UK 2Biochemiezentrum der Universität Heidelberg (BZH), Heidelberg, Germany *Corresponding author. School of Biological Sciences, University of Manchester, Smith Building, Oxford Road, Manchester M13 9PT, UK. Tel.: +44 161 275 5070; Fax: +44 161 275 5082; E-mail: [email protected] The EMBO Journal (2004)23:2755-2764https://doi.org/10.1038/sj.emboj.7600281 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo. Introduction Tail-anchored (TA) proteins display their hydrophilic N-termini to the cytosol and are widely distributed throughout the membranes of eukaryotic organelles (Wattenberg and Lithgow, 2001; Borgese et al, 2003). Their principal sites of membrane integration are the endoplasmic reticulum (ER) and mitochondrial outer membrane (Kutay et al, 1995; Borgese et al, 2001), and they ultimately function in many key cellular processes including vesicle trafficking (SNAREs), protein translocation (Sec61β and Sec61γ) and apoptosis (Bcl-2 family). The C-terminal location of the sole ER-targeting signal present in TA proteins poses a unique challenge to the cellular targeting and membrane integration machineries, since it only emerges from the ribosome and becomes available to cytosolic targeting factors post-translationally (Kutay et al, 1995). At the ER membrane, authentic integration of TA proteins is strictly dependent upon proteinaceous factors (Kutay et al, 1995), although their precise identity remains controversial and incompletely defined (Kutay et al, 1995; Steel et al, 2002; Abell et al, 2003; Yabal et al, 2003). The post-translational integration of TA proteins in vitro is ATP dependent, consistent with a role of cytosolic chaperones in maintaining the precursor in an insertion-competent state (Kutay et al, 1995; Kim et al, 1999; Steel et al, 2002). However, no cytosolic components responsible for the organelle-specific targeting of TA proteins have been identified to date (Kutay et al, 1995; Kim et al, 1999; Borgese et al, 2001). The in vitro integration of an archetypal TA protein, cytochrome b5 (Cytb5), can occur independently of the signal recognition particle (SRP), consistent with a post-translational membrane insertion pathway (Anderson et al, 1983). In contrast, the insertion of most membrane proteins at the ER depends upon SRP binding to their loosely conserved N-terminal signal sequences and internal signal anchors. A key feature of these signals is a contiguous stretch of 7–15 hydrophobic residues that interacts with the 54 kDa subunit of SRP (SRP54) (Keenan et al, 2001). Furthermore, SRP specifically associates with the ribosome at the exit tunnel, thereby allowing it to bind signal sequences immediately upon their emergence (Pool et al, 2002; Halic et al, 2004). Once SRP has bound a signal sequence, it promotes cotranslational membrane insertion by attenuating translation until the ribosome-nascent chain complex (RNC) has been received by the SRP receptor (Egea et al, 2004; Focia et al, 2004) at the ER membrane and transferred to the Sec61 protein translocation channel (Song et al, 2000). The strictly cotranslational nature of SRP action is supported by the observation that SRP dissociates completely from nascent secretory proteins immediately upon their release from the ribosome (Wiedmann et al, 1994; Plath and Rapoport, 2000). In order to identify cellular factors responsible for the ER targeting of TA proteins, we pursued a crosslinking approach using polypeptides that had been synthesised in reticulocyte lysate. Given previous studies (Anderson et al, 1983; Kutay et al, 1995), we were initially surprised to discover that TA proteins form adducts with SRP54. However, using the well-characterised ER-targeted TA proteins synaptobrevin 2 (Syb2) and Sec61β, we show that SRP can specifically associate with TA proteins in a strictly post-translational manner, and via an interaction that is mediated by the proteins' hydrophobic insertion sequences. At a functional level, we find that TA proteins can be targeted to ER membranes via a previously undefined post-translational pathway that is dependent upon SRP, the SRP receptor and GTP. The efficient operation of this pathway requires the presence of SRP during nascent chain release from the ribosome, but ribosomes play no role in the targeting of the polypeptide to the ER membrane. We find that the capacity to utilise this SRP-dependent route is precursor specific, and, while this pathway accounts for a significant proportion of Syb2 and Sec61β integration, Cytb5 insertion is entirely independent of this route. On the basis of these findings, we propose a unifying model to describe the pathways responsible for TA protein biosynthesis in vivo. Results TA proteins were synthesised in rabbit reticulocyte lysate using mRNAs lacking stop codons, thereby enabling the release of the polypeptides from the ribosome to be synchronised by puromycin treatment (Gilmore et al, 1991). Associations between the newly released TA proteins and cytosolic factors present in the complete cell-free system were then assessed by crosslinking. As a benchmark for SRP binding, we used the N-terminal 86 residues of the secretory protein preprolactin (PPL86) in its cycloheximide-stabilised, ribosome-bound, form. Consistent with previous studies (Wiedmann et al, 1994), SRP54 was one of several cytosolic components crosslinked to PPL86 (Figure 1A, lanes 1–3). Surprisingly, Syb2 chains that had been released from the ribosome by puromycin treatment also crosslinked to SRP54 (Figure 1A, lanes 5–7), as did puromycin-released Sec61β chains (Figure 1A, lanes 10–12). Hence, TA proteins can associate with the SRP54 subunit in a post-translational manner. In contrast, we found no evidence of crosslinking between these TA proteins and members of the Hsc70 chaperone family (data not shown), although these components are estimated to be present at a 500-fold molar excess over SRP (Siegel and Walter, 1988; Nollen et al, 2000). Several of the other adducts observed during this analysis were found to reflect in vitro ubiquitination of the precursor proteins (Figure 1A, lanes 4, 8 and 13), as previously seen during studies of preproα-factor (Plath and Rapoport, 2000). Figure 1.TA proteins associate with SRP54 post-translationally. (A) Stop codon minus RNA encoding full-length Syb2 (lanes 5–9), full-length Sec61β (lanes 10–14) or the first 86 residues of preprolactin (lanes 1–4) was translated for 20 min, and synthesis then terminated by the addition of cycloheximide (CHX) or puromycin (puro) as shown. Crosslinking was induced with DSS as indicated. Total products were analysed directly (lanes 1, 2, 5, 6, 10 and 11) or following immunoprecipitation with antisera for SRP54 (αSRP54), ubiquitin (αUb) or a nonrelated serum (NRS). (B) Amino-acid sequences of the hydrophobic ER-targeting signals from the polypeptides used in this study. Potential TM domains are underlined, while dots indicate regions where hydrophilic domains of the polypeptide extend beyond the sequence presented. Numbers in superscript show the total length of the various polypeptides used. Where two numbers are given, the first is the limit of the sequence presented and the second the length of the polypeptide studied. (C) Cell-free translations of the various precursors were terminated with puromycin and the samples treated with DSS. In order to avoid any variability resulting from differences in translation efficiency, a fraction of each sample was first analysed by SDS–PAGE and the relative amount of each nascent chain determined by quantitative phosphorimaging (data not shown). On the basis of this analysis, equivalent amounts of each DSS-treated radiolabelled precursor were then used for the immunoprecipitation of adducts with SRP54. (D) Stop codon minus RNA was translated for 20 min in the presence or absence of canine pancreatic microsomes, and then incubated with puromycin for a further 20 min to enable membrane integration. The membrane fraction was isolated by centrifugation through a high-salt sucrose cushion. The resulting pellet was then resuspended in alkaline sodium carbonate solution and the membrane pellet re-isolated by centrifugation. For each precursor, the translation reaction lacking any exogenously added membranes was processed in parallel to provide an estimate of the amount of each protein that was recovered in the final pellet fraction independent of membrane integration. The amount of precursor present in each of the fractions was determined by SDS–PAGE and quantitative phosphorimaging, and was expressed as a percentage of the total protein synthesised in that reaction. The final value for membrane integration is the percentage of the total protein synthesised, which was specifically recovered with the isolated microsomal membranes (see Supplementary data). These values are the means of two or more independent experiments, and standard errors are indicated. Download figure Download PowerPoint SRP54 normally binds to the hydrophobic core of ER signal sequences (Keenan et al, 2001), and we therefore investigated the validity of the interaction between SRP54 and the TA proteins by manipulating their hydrophobic tail anchors (see Figure 1B). When the Syb2 tail anchor is truncated from 20 to 10 residues (Syb2-0.5TM), its association with SRP54 is substantially reduced, while upon its complete removal (Syb2-TM) no association with SRP54 is detected (Figure 1C, cf. lanes 1–3). The hydrophobic core from the PPL signal sequence could replace the authentic Syb2 tail anchor (Syb2:PPL) and facilitate a strong interaction with SRP54 (Figure 1C, lane 4). However, a nonfunctional version of the PPL signal sequence (Syb2:PPL.KO; see Luirink et al, 1992) gave no adducts with SRP54 (Figure 1C, lane 5). When the tail-anchor regions of Syb2 and Sec61β were replaced with hydrophilic sequences (Syb2:GC and Sec61β:GC, respectively), no crosslinking to SRP54 was observed (Figure 1C, lanes 6 and 8). Thus, the binding of cytosolic SRP to TA proteins requires an intact, hydrophobic, ER-targeting signal. The efficacy of the crosslinking to SRP54 observed in this assay was further investigated by analysing the PPL86 polypeptide after puromycin treatment to release it from the ribosome. In agreement with previous studies (Wiedmann et al, 1987), no adducts of PPL86 with SRP54 were observed (Figure 1C, lane 9; cf. Figure 1A, lane 3), confirming the efficiency of the puromycin release and the specificity of adduct formation between SRP54 and the TA proteins. If the binding of SRP to the C-terminal hydrophobic targeting sequences of TA proteins was functionally significant, a defect in SRP binding should result in a defect in membrane integration, and we therefore investigated this possibility. Ribosome-bound versions of the polypeptides were generated in the presence of canine pancreatic microsomes, and the precursors then released from the ribosome by puromycin treatment. Parallel control reactions were carried out in the absence of any membranes, and integration was quantitatively assessed by determining the resistance of the membrane-associated precursor proteins to extraction with alkaline sodium carbonate solution (see Kutay et al, 1995; Whitley et al, 1996; cf. Figure 4A). As previously observed (cf. Kutay et al, 1995; Whitley et al, 1996), when no membranes were present in the incubations, only background levels of the TA precursors were recovered in the final pellet (see Supplementary data). Likewise, precursors with disrupted or deleted TA sequences showed very low levels of membrane integration (Figure 1D). In contrast, authentic TA proteins that could recruit SRP (Figure 1C) were efficiently membrane integrated (e.g. 27% of Syb2 and 48% of Sec61β; see Figure 1D). Given the role of the TA sequence in mediating stable membrane insertion, the reductions in integration observed upon perturbation of the hydrophobic TA sequences are likely to reflect defects in both targeting and membrane insertion. Nevertheless, taken together, these data are consistent with the hypothesis that the binding of SRP to TA proteins might play an important role in their delivery to the ER membrane. We examined the precise relationship between SRP binding and the state of the TA polypeptide, focusing on the question of whether the interaction was strictly post-translational. To this end, we analysed the association of SRP with Syb2 when it was present as either an artificially generated ribosome/nascent chain complex (RNC) or a fully released polypeptide. The ribosome attachment of the nascent chain was specifically monitored by analysing the crosslinking of Syb2 to NACα, a component that is known to interact with nascent chains in a sequence-independent, but strictly ribosome-dependent, manner (Wiedmann et al, 1994). In the case of PPL86, the bulk of the nascent chains and all their adducts with both SRP54 and NACα co-isolated with the ribosomal pellet after stabilisation with cycloheximide (Figure 2, lane 2). After puromycin treatment, the PPL86 nascent chains were recovered primarily in the supernatant, and adducts with both SRP54 and NACα were absent (Figure 2, lane 3). Hence, PPL86 will only associate with SRP54 and NACα as a nascent, ribosome-attached, polypeptide chain (Wiedmann et al, 1994; Plath and Rapoport, 2000). Syb2 RNCs responded to both cycloheximide and puromycin treatment in the same fashion as PPL86 RNCs (Figure 2, Syb2 nascent chain, lanes 1–4), and the Syb2-NACα adducts were similarly restricted to the ribosome-attached form of the polypeptides (Figure 2, lane 2). In contrast, the Syb2 adduct with SRP54 was seen exclusively in the ribosome-free supernatant and primarily after puromycin release (Figure 2, lane 3). A small fraction of Syb2 chains were lost from the ribosome even with cycloheximide stabilisation (Figure 2, Syb 2 nascent chain, cf. lanes 1 and 2), resulting in the appearance of some SRP54 adducts under these conditions (Syb2, αSRP54, lane 1). We conclude that SRP binds to TA proteins in a strictly post-translational manner. Figure 2.TA protein association with SRP54 is strictly post-translational. Syb2 or PPL86 RNA was translated for 5 min, treated with cycloheximide or puromycin, and crosslinked using DSS. RNCs were pelleted through a high-salt cushion (pel), leaving free polypeptides in the supernatant (sup). A fraction of each sample was analysed prior to crosslinking (PPL85 and Syb2), while the remainder was used for the immunoprecipitation of adducts with NACα and SRP54 as indicated. Download figure Download PowerPoint To determine the region of the TA protein that binds to SRP54, we performed a site-specific crosslinking analysis using four precursor proteins that each contains a single, naturally occurring, cysteine residue within its hydrophobic tail-anchor sequence. When crosslinking was mediated by a short (6 Å), bifunctional, cysteine reactive reagent (o-PDM), distinct adducts with SRP54 were detected using both the full-length proteins (Figure 3, lanes 1 and 3, o-PDM), or their hydrophobic tail-anchor regions alone (Figure 3, lanes 5 and 7, o-PDM). As an additional control, point mutants of each of these four polypeptides, where the single cysteine was altered to a leucine residue, were analysed in parallel. In this case, no significant o-PDM-dependent crosslinking to SRP54 was detected (Figure 3, lanes 2, 4, 6 and 8), confirming the authenticity of the adducts seen with the cysteine-containing polypeptides. In some cases, trace levels of o-PDM-dependent adducts were obtained with derivatives lacking any cysteine residues (Figure 3, lanes 4 and 8). This probably reflects the low-efficiency modification of amino groups by o-PDM. When the same series of polypeptides was analysed using a bifunctional crosslinking reagent specific for the several amino groups present in each polypeptide, adducts with SRP54 were observed with all of the precursors (Figure 3, lanes 1–8, DSS). Taken together, the ability of SRP54 to be crosslinked to a single cysteine residue located near the centre of their ER-targeting signals strongly suggests that SRP associates directly with the hydrophobic regions of TA proteins. Figure 3.SRP associates with the hydrophobic domain of TA proteins. Stop codon minus RNAs encoding the indicated polypeptides with either a single cysteine probe (upper panel) or a leucine at the equivalent location (lower panel) was translated for 15 min. The resulting RNCs were then isolated through a low-salt cushion to separate the precursors from DTT in the translation reaction and facilitate crosslinking with the bifunctional maleimide o-PDM. Nascent chains were released from ribosomes with puromycin, treated with DSS or o-PDM, and adducts with SRP54 recovered by immunoprecipitation. The relative efficiency with which each precursor was synthesised was established by quantitative phophorimaging prior to immunoprecipitation, and equivalent amounts of radiolabelled polypeptides were used for subsequent immunoisolation as for Figure 1C. Download figure Download PowerPoint We next investigated the biological significance of the specific interaction between SRP and TA proteins that we had defined. A hallmark of the SRP-dependent targeting pathway is its dependency upon GTP (Keenan et al, 2001). This GTP requirement can be substituted with nonhydrolysable analogues such as GMPPNP in the context of typical in vitro assays, where a single round of SRP-dependent targeting is normally sufficient to promote significant membrane integration (Connolly et al, 1991; High et al, 1991). Under these conditions, the lack of SRP recycling from the ER membrane that results from the absence of GTP hydrolysis has little effect (Connolly et al, 1991). As a read-out for protein targeting, we determined the efficiency of Syb2 membrane integration (cf. Figure 1D), in this case following various pretreatments of the cell-free translation reaction (outlined in Figure 4A). In the control reaction, we found that ∼7% of total Syb2 was specifically integrated into ER membranes during a short, 5 min, post-treatment incubation (Figure 4B, cf. lanes 1 and 2, upper panel). This level of efficiency is on a par with previous studies where quantification has been carried out (Whitley et al, 1996; Kim et al, 1999; Lan et al, 2000), and reflects the relatively short incubation period used for this insertion assay (cf. Figure 1D, where a 20 min incubation was used). As previously observed in other studies, the substantial depletion of NTPs from the cell-free system prior to membrane addition largely abolished the post-translational integration of Syb2 into ER-derived membranes (Figure 4B, cf. lanes 2 and 4, upper panel; see Kutay et al, 1995). However, we made the striking observation that the nonhydrolysable GTP analogue GMPPNP could restore Syb2 membrane integration to 42% of the original level (Figure 4B, cf. lanes 5 and 6, upper panel). In contrast, the addition of the ATP analogue AMPPNP caused no increase in membrane integration (Figure 4B, lanes 7 and 8, upper panel), confirming that the stimulation was specific for GTP analogues. Likewise, a combination of GMPPNP and AMPPNP gave the same stimulation as GMPPNP alone (Figure 4B, cf. lanes 5 and 6 with 9 and 10, upper panel). If the addition of GMPPNP were enabling a biologically productive role for SRP in promoting the authentic membrane integration of Syb2, one would expect a reciprocal release of SRP from the TA proteins upon the addition of ER membranes bearing the SRP receptor. Consistent with this proposal, we find that the addition of GMPPNP and membranes results in a significant reduction of Syb2-SRP54 adduct formation to a level that mirrors the stimulation of membrane insertion observed (Figure 4B, lanes 3–6, 9 and 10, lower panel). As with integration, the addition of AMPPNP in combination with microsomal membranes causes no significant reduction in SRP54 adduct formation. We conclude that Syb2 can become membrane integrated via a GTP-dependent pathway that correlates with the release of SRP from the polypeptide. To further investigate the role of SRP in the targeting of TA proteins, we manipulated the SRP receptor (SR), which is sensitive to low concentrations of trypsin that specifically remove the SRα subunit but leave the SRβ subunit largely intact (Andrews et al, 1989; Miller et al, 1995). In this case, we found that ∼8% of the total Syb2 present in the incubation was specifically membrane integrated in the control reaction (Figure 5A, cf. lanes 1 and 2). If the ER-derived membranes were first treated with trypsin, we found that their capacity for integrating Syb2 was reduced to 23% of the original level, consistent with a role for specific ER proteins in mediating targeting/integration (Figure 5A, lane 4; cf. Kutay et al, 1995). However, when these trypsinised membranes were specifically reconstituted with recombinant SR (Fulga et al, 2001), a substantial recovery of Syb2 membrane integration to 65% of the original level was obtained (Figure 5A, lane 5). In contrast, the addition of recombinant SR in the absence of trypsin treatment had only a marginal effect on Syb2 integration (Figure 5A, lanes 2 and 3). To confirm the specificity of this reconstitution assay, we determined the efficiency of targeting of the α subunit of SR (SRα). SRα is a peripheral ER membrane protein that binds directly to the integral membrane β subunit via a mechanism that is independent of SRP (Andrews et al, 1989). In contrast to the effect upon Syb2 integration, SRα was relatively unaffected by trypsin treatment and SR reconstitution, with ∼7% of the total input being specifically recovered with the membrane fraction under all conditions (Figure 5B, cf. lanes 2–5). Figure 4.Membrane insertion of Syb2 is GTP dependent. (A) Flow diagram of the assay used to investigate the nucleotide dependence of Syb2 integration following apyrase treatment. (B) Syb2 RNA was translated for 20 min and treated with puromycin. Samples were depleted of nucleotide di- and triphosphates by treatment with apyrase, or mock treated. Aliquots were supplemented with combinations of AMPPNP, GMPPNP and RMs. After a 5 min incubation, the extent of membrane integration was assessed using sodium carbonate extraction, and SRP release assessed by DSS crosslinking and immunoprecipitation with αSRP54. Relative membrane integration (see the upper panel) was calculated by quantifying the increase in sodium carbonate-resistant material upon the addition of RMs (+), relative to a control sample with no added RMs (−), cf. Supplementary data. Mock-treated membranes were set at 100% relative membrane integration (lane 2). SRP release (lower panel) indicates the reduction in the amount of SRP54 adduct caused by the addition of RMs. Download figure Download PowerPoint Figure 5.Membrane insertion of Syb2 is dependent on the SRP receptor. (A) Syb2 RNA lacking a stop codon was translated for 20 min and then treated with puromycin. RMs were treated with trypsin or mock treated as indicated, re-isolated by centrifugation, and added to the translation mix with or without soluble SR. After a 5 min incubation, membrane integration was assessed by resistance to sodium carbonate extraction. Samples were separated by SDS–PAGE. Quantification of membrane integration was performed as described for Figure 4B, with mock-treated RM set to 100%. Lane 1 shows the level of background signal obtained in the absence of any added membranes. This contribution was subtracted from the signals obtained in the presence of membranes during quantification, and is therefore labelled as 0% relative integration. (B) SRα RNA was translated in vitro and the membrane association of the resulting protein analysed as described in (A). (C) The membrane integration of Syb2 into various RM preparations was performed as described in (A), except that the RMs were reconstituted with in vitro-synthesised SRα in place of the recombinant protein used in panels A and B. SRP54 release indicates the percentage reduction in SRP54 adduct formation caused by the addition of the various RM preparations. In this case, the mean and standard error of two independent experiments are shown. (D) The membrane integration of Syb2G into various RM preparations was established as described in (C), except that the level of N-glycosylation was assayed in place of the amount of material resistant to sodium carbonate extraction. (E) The membrane integration of Sec61β was determined as described in (C). (F) The membrane integration of Cytb5C was determined as described in (C). Download figure Download PowerPoint In vitro-synthesised SRα is also known to rescue the targeting defect resulting from the trypsin treatment of ER membranes (Andrews et al, 1989). As with the recombinant protein, we found that the addition of in vitro-synthesised SRα resulted in a significant recovery of specific membrane integration after trypsinisation (20–64%; Figure 5C). This increase in membrane integration was accompanied by a comparable increase in the release of SRP from the Syb2 polypeptides, as judged by the accompanying reduction in its crosslinking to the SRP54 subunit (21–71%; Figure 5C). To further verify that we were observing an SR-dependent stimulation in authentic membrane insertion, we exploited a derivative of Syb2 with a site for N-linked glycosylation at its C-terminus (Syb2G; see Figure 1B). The efficiency of Syb2G N-glycosylation (∼5% of total input) was comparable to that seen in previous studies (Kutay et al, 1995), and we used the quantity of glycosylated product as a specific measure of complete membrane insertion. As with Syb2, we found that, when trypsinised pancreatic microsomes were repopulated with in vitro synthesised SRα, an ∼40% recovery in the levels of N-glycosylated material was obtained (Figure 5D, cf. tryp and tryp plus SRα samples). Hence, Syb2G responds to the re-addition of SRα in the same manner as native Syb2, and we conclude that SR stimulates the topologically correct insertion of Syb2 at the ER membrane. In order to establish whether the SR-dependent integration of TA proteins was a general effect, we investigated two other precursors, Sec61β, which we had found to crosslink SRP54 (Figure 1A), and a point mutant of cytochrome b5 (Cytb5C), which can integrate into the ER independently of SRP (Anderson et al, 1983). In the case of Sec61β, while the residual integration observed after trypsin treatment was higher than that for Syb2 or Syb2G (cf. Figure 5C–E), an ∼30% stimulation in membrane integration was observed upon repopulation of trypsinised membranes with in vitro synthesised SRα (Figure 5E). In contrast, the integration of Cytb5C was essentially unaffected by either trypsinisation or subsequent re-addition of SRα (Figure 5F). We therefore conclude that some TA proteins can be membrane integrated in an SR-dependent manner,