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• W1988625335 abstract "Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion. Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion. Membrane protein insertion at the mammalian ER occurs most commonly via the cotranslational pathway, in which a hydrophobic signal sequence emerges from the ribosome and is recognized by the signal recognition particle (SRP) 1The abbreviations used are: SRP, signal recognition particle; BMH, bismaleimidohexane; ER, endoplasmic reticulum; Ii, invariant chain; SPC25, 25-kDa subunit of the signal peptidase complex; Syb2, synaptobrevin 2; Syb2cm, cysteine mutant of synaptobrevin 2; Syn1A, syntaxin 1A 1The abbreviations used are: SRP, signal recognition particle; BMH, bismaleimidohexane; ER, endoplasmic reticulum; Ii, invariant chain; SPC25, 25-kDa subunit of the signal peptidase complex; Syb2, synaptobrevin 2; Syb2cm, cysteine mutant of synaptobrevin 2; Syn1A, syntaxin 1A(1Meacock S.L. Greenfield J.J.A. High S. Essays Biochem. 2000; 36: 1-13Google Scholar). The ribosome-nascent chain-SRP complex is then targeted to the ER membrane via an association with a cognate receptor complex (1Meacock S.L. Greenfield J.J.A. High S. Essays Biochem. 2000; 36: 1-13Google Scholar, 2Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Google Scholar). Upon its arrival at the ER, the nascent membrane protein is delivered to the Sec61 translocon. This comprises multiple Sec61 heterotrimers, composed of α, β, and γ subunits, and it functions as the ER membrane insertion site for precursors delivered via the SRP-dependent targeting route (1Meacock S.L. Greenfield J.J.A. High S. Essays Biochem. 2000; 36: 1-13Google Scholar, 2Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Google Scholar, 3High S. Laird V. Trends Cell Biol. 1997; 7: 206-210Google Scholar, 4Matlack K.E. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Google Scholar). It is noteworthy that in higher eukaryotes, the membrane insertion of proteins targeted via the SRP-dependent pathway appears to be principally cotranslational, with the ribosome remaining closely associated with the Sec61 translocon during membrane integration (5Menetret J.F. Neuhof A. Morgan D.G. Plath K. Radermacher M. Rapoport T.A. Akey C.W. Mol. Cell. 2000; 6: 1219-1232Google Scholar, 6Beckmann R. Spahn C.M. Eswar N. Helmers J. Penczek P.A. Sali A. Frank J. Blobel G. Cell. 2001; 107: 361-372Google Scholar). In contrast to higher eukaryotes, SRP-independent, post-translational translocation plays a significant role in the yeast Saccharomyces cerevisiae (7Zheng N. Gierasch L.M. Cell. 1996; 86: 849-852Google Scholar, 8Ng D.T.W. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Google Scholar). In this instance the precursors use cytosolic chaperones to maintain translocation competence (9Plath K. Rapoport T.A. J. Cell Biol. 2000; 151: 167-178Google Scholar), and signal sequence recognition occurs at the Sec complex of the ER membrane (10Brodsky J.L. Trends Biochem. Sci. 1996; 21: 122-126Google Scholar,11Plath K. Mothes W. Wilkinson B.M. Stirling C.J. Rapoport T.A. Cell. 1998; 94: 795-807Google Scholar). This Sec complex is made up of the heterotrimeric Sec61 complex together with four other membrane-associated components, namely Sec62p, Sec63p, Sec71p and Sec72p, as well as the ER luminal chaperone Kar2p (the S. cerevisiae equivalent of BiP) (12Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Google Scholar, 13Lyman S.K. Schekman R. Cell. 1997; 88: 85-96Google Scholar, 14Matlack K.E. Misselwitz B. Plath K. Rapoport T.A. Cell. 1999; 97: 553-564Google Scholar). It has become apparent that Sec62p and Sec63p are not restricted to S. cerevisiae, and similar proteins have been identified in mammals, although their precise function is unknown (15Meyer H.A. Grau H. Kraft R. Kostka S. Prehn S. Kalies K.U. Hartmann E. J. Biol. Chem. 2000; 275: 14550-14557Google Scholar, 16Tyedmers J. Lerner M. Bies C. Dudek J. Skowronek M.H. Haas I.G. Heim N. Nastainczyk W. Volkmer J. Zimmermann R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7214-7219Google Scholar). Specific examples of post-translational translocation have been identified in higher eukaryotes, although these tend to be the exception rather than the rule. In the case of very short presecretory proteins, such as prepromelittin, the N-terminal signal sequence does not have an opportunity to interact with SRP before translation is terminated (17Schlenstedt G. Zimmermann R. EMBO J. 1987; 6: 699-703Google Scholar,18Müller G. Zimmermann R. EMBO J. 1987; 6: 2099-2107Google Scholar). Hence, prepromelittin translocation is independent of SRP but dependent upon cytosolic component(s) and ATP, presumably to maintain the polypeptide in a “translocation-competent” state (18Müller G. Zimmermann R. EMBO J. 1987; 6: 2099-2107Google Scholar). Tail-anchored proteins form a distinct class of integral membrane proteins, possessing a single membrane insertion sequence at their C terminus and displaying their remaining N-terminal portion in the cytosol. The majority of tail-anchored proteins become integrated at the ER membrane (19–21, but see also Ref. 22Borgese N. Gazzoni I. Barberi M. Colombo S. Pedrazzini E. Mol. Biol. Cell. 2001; 12: 2482-2496Google Scholar), and members of this class carry out a range of important cellular functions, such as ER translocation (Sec61β and Sec61γ), vesicle recognition (solubleN-ethylmaleimide-sensitive factor attachment protein receptor, or SNARE proteins), and electron transfer (cytochromeb 5) (for reviews, see Refs. 23Kutay U. Hartmann E. Rapoport T.A. Trends Cell Biol. 1993; 3: 72-75Google Scholar and 24Wattenberg B. Lithgow T. Traffic. 2001; 2: 66-71Google Scholar). In the case of synaptobrevin 2 (Syb2), the authentic membrane insertion sequence can be replaced by a polyleucine span with a minimum length of 12 residues, demonstrating the lack of any specific sequence requirements for this region (25Whitley P. Grahn E. Kutay U. Rapoport T.A. von Heijne G. J. Biol. Chem. 1996; 271: 7583-7586Google Scholar). The membrane insertion sequence of tail-anchored proteins acts as an ER targeting signal, and its relative position dictates that membrane insertion is post-translational and hence likely to be SRP-independent (9Plath K. Rapoport T.A. J. Cell Biol. 2000; 151: 167-178Google Scholar, 26Anderson D.J. Mostov K.E. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7249-7253Google Scholar). To date, only general characteristics regarding the targeting and membrane insertion of tail-anchored proteins are known. The process is ATP-dependent, consistent with a role for cytosolic chaperones in maintaining the polypeptides in an integration-competent state (19Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Google Scholar, 20Linstedt A.D. Foguet M. Renz M. Seelig H.P. Glick B.S. Hauri H.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5102-5105Google Scholar, 21Behrens T.W. Kearns G.M. Rivard J.J. Bernstein H.D. Yewdell J.W. Staudt L.M. J. Biol. Chem. 1996; 271: 23528-23534Google Scholar, 27Zimmermann R. Sagstetter M. Lewis M.L. Pelham H.R.B. EMBO J. 1988; 7: 2875-2880Google Scholar, 28Kim P.K. Janiak-Spens F. Trimble W.S. Leber B. Andrews D.W. Biochemistry. 1997; 36: 8873-8882Google Scholar, 29Kim P.K. Hollerbach C. Trimble W.S. Leber B. Andrews D.W. J. Biol. Chem. 1999; 274: 36876-36882Google Scholar). Furthermore, the membrane insertion of tail-anchored proteins includes a membrane binding step that is saturable (29Kim P.K. Hollerbach C. Trimble W.S. Leber B. Andrews D.W. J. Biol. Chem. 1999; 274: 36876-36882Google Scholar), and this process is also sensitive to prior treatment of the membranes with protease (19Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Google Scholar). Although both of these observations suggest that the membrane insertion of tail-anchored polypeptides is mediated by proteins, the ER components responsible for this process have remained unidentified. We set out to identify ER components that may mediate the membrane insertion of tail-anchored proteins using a defined cross-linking approach to identify proteins that are transiently associated with the newly membrane-integrated polypeptides. We report a defined sequence of associations between newly made tail-anchored proteins and both the Sec61 translocon and Sec61 translocon-associated components. Most significantly, we observe a similar sequence of events with a comparable, cotranslationally inserted, signal-anchored protein. We conclude that the biosynthesis of tail-anchored and signal-anchored proteins is mediated by a similar complement of ER components. Polyclonal antibodies were raised against specific peptides representing Sec61α, Sec61β (from B. Dobberstein), SPC25, Sec62p, and Sec63p. The anti-Syb2 antibody was a mouse monoclonal recognizing an epitope at the N terminus of Syb2 (from R. Jahn). An NcoI fragment incorporating the coding region of the human Sec61β cDNA (30Hartmann E. Sommer T. Prehn S. Görlich D. Jentsch S. Rapoport T.A. Nature. 1994; 367: 654-657Google Scholar) was subcloned into the pSPUTK vector (Stratagene). The DNA template for its transcription was made by cleavage of the Sec61β pSPUTK plasmid with EcoRI. The cDNA for rat Syb2 in pBluescript was a gift from R. Scheller (Stanford University). The cysteine mutant of synaptobrevin (Syb2cm) was created by changing a leucine (Leu-63) to a cysteine using the QuikChange site-directed mutagenesis kit (Stratagene). The following sense primer was used: 5′-GACCAGAAGCTATCGGAATGCGATGATCGCGCAGATGCCC-3′, and the resulting plasmid was linearized for transcription usingXbaI. The cDNA for rat Syn1A was a gift from Dr. Sabine Hilfiker (University of Manchester). The coding region was amplified by PCR, introducing a BglII site 1 base 5′ to the initiation codon using primer 5′-ACTTTGGCAGATCTACCATGAAGGACCGAACCCAGG-3′ and anXbaI site after the termination codon using primer 5′-CACCATCGGGGGCATCTTTGGATAGTCTAGATATA-3′. This PCR product was cut with BglII and ligated into the BglII andHpaI sites of pSPUTK (Stratagene). The resulting plasmid was linearized for transcription using XbaI. The coding region of invariant chain (Ii) was amplified by PCR using the forward primer 5′-ACTTTGGCAGATCTACCATGGATGACCAGCGCGACC-3′ to introduceBglII and NcoI sites around the initiation codon and replace the asparagine residue at position 3 of the coding region with a cysteine. An XbaI site was introduced after the termination codon by using the reverse primer 5′-TATATCTAGATCACATGGGGACTGGGCC-3′. This PCR product was cut withBglII and ligated into the BglII andHpaI sites of pSPUTK (Stratagene). The natural cysteine (Cys-28) was changed to a methionine using the QuikChange kit with the sense primer 5′-CCGGAGAGCAAGATGAGTCGCGGAGCCCTG-3′. Transcription templates for Ii derivatives were prepared by PCR using this variant of Ii as a template. The 5′-primer recognized a region 150 bp 5′ to the SP6 promoter 5′-CCAGAAACTCAGAAGGTTCG-3′, whereas the 3′ primers were 5′-TATATCAGTACAGGAAGTAGGCGGTGG-3′ for Ii81 and 5′-CATGGGGACTGGGCCCAG-3′ for IiTA. The authenticity of all PCR-derived constructs was confirmed by DNA sequencing. Transcripts were synthesized using T3 RNA polymerase for Syb2cm, or SP6 RNA polymerase for all the other templates, according to the manufacturer's instructions (New England Biolabs). Proteins were synthesized using rabbit reticulocyte lysate (Promega) that had been prespun at 200,000 × g for 10 min to remove any contaminating membranes. Incubations were performed at 30 °C in the presence of both [35S]methionine and canine pancreatic microsomes according to the manufacturer's instructions. Microsomes were prepared from canine pancreas as described by Walter and Blobel (31Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Google Scholar) and added to in vitro translations at 1.5–2.0A 280/ml. A time course was performed to establish the amount of Sec61β that was integrated into canine pancreatic microsomes under the experimental conditions that we were using. This analysis was carried out across a 60-min period, and the percentage integration was defined as the proportion of the total protein synthesized at a particular time point which was found to be membrane-associated and resistant to extraction with alkaline sodium carbonate solution (23Kutay U. Hartmann E. Rapoport T.A. Trends Cell Biol. 1993; 3: 72-75Google Scholar, 32Knight B.C. High S. Biochem. J. 1998; 331: 161-167Google Scholar). Control experiments showed that in the absence of added microsomes, or when using a protein that lacked its tail anchor sequence, less than 2% of the total protein synthesized in 30 min was recovered by this assay (data not shown). In contrast, >30% of authentic Sec61β was recovered in the membrane fraction using the same assay (see Fig.2 A). On the basis of this analysis, translation reactions were initially carried out for 30 min by which point significant membrane integration was observed (see Fig. 2 A). In some subsequent experiments, shorter incubation periods were used to optimize transient associations. In these cases, specific details are indicated in the accompanying figure legend. All puromycin treatments were performed by the addition of 1 mm puromycin and subsequent incubation at 30 °C for 5 min. Microsomes were isolated for cross-linking analysis by layering them over HSC buffer (250 mm sucrose, 500 mm KOAc, 5 mmMg(OAc)2, 50 mm Hepes-KOH pH 7.9) and spinning at 100,000 × g for 10 min to yield a membrane pellet. Membrane pellets were resuspended in LSC buffer (250 mmsucrose, 100 mm KOAc, 5 mm(MgOAc)2, 50 mm Hepes-KOH pH 7.9) and normally incubated immediately at 30 °C for 10 min with either bismaleimidohexane (BMH) (0.5 mm final concentration unless otherwise stated), diluted from a 20 mm stock dissolved in dimethyl sulfoxide, or an equivalent dimethyl sulfoxide control. Where time course experiments were carried out, the sample was resuspended in LSC buffer and incubated at 30 °C for 0–120 min before cross-linking as above. In one case a parallel sample was incubated at 0 °C for 60 min before adding BMH. Cross-linking was stopped by the addition of 10 mm 2-mercaptoethanol to quench any unreacted maleimide groups, and a fraction of the sample was removed for direct analysis. The remainder of the samples were denatured at 70 °C for 10 min in the presence of 1% (w/v) SDS and diluted with 4 volumes of Triton X-100 immunoprecipitation buffer (1% (w/v) Triton X-100, 140 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 7.5). Samples were precleared by the addition of 10% volume Pansorbin (Calbiochem) and incubated at 4 °C for 1 h, followed by centrifugation at 15,000 × g for 5 min. The resulting supernatants were subjected to immunoprecipitation by the addition of antisera at 1:100 (v/v) and incubation at 4 °C for 16 h with mixing. Protein A-Sepharose was added to 10% volume, and the incubation continued at 4 °C for a further 2 h. The protein A-Sepharose beads and bound material were pelleted in a microfuge and washed several times with Triton X-100 immunoprecipitation buffer. The resulting beads were heated to 70 °C for 10 min in SDS-PAGE sample buffer, and unless otherwise stated, the solubilized material was resolved on 16% polyacrylamide Tris-glycine gels run under denaturing conditions. We chose to investigate the association of newly synthesized tail-anchored proteins with ER components using the bifunctional cross-linking reagent BMH, which is highly specific for cysteine residues. This approach had proven very efficient in previous studies of cotranslational membrane protein biosynthesis (32Knight B.C. High S. Biochem. J. 1998; 331: 161-167Google Scholar, 33Laird V. High S. J. Biol. Chem. 1997; 272: 1983-1989Google Scholar). Furthermore, our preliminary analysis had shown that the membrane insertion of the tail-anchored proteins into ER-derived microsomes could be prevented by prior treatment of the membranes withN-ethylmaleimide, which modifies the free sulfydryl groups of cysteine residues (data not shown). Hence, BMH was ideally suited to cross-link newly synthesized tail-anchored proteins containing one or more cysteine residues (see Fig. 1) to the N-ethylmaleimide-sensitive, cysteine-containing ER proteins that facilitate their membrane insertion. Given our interest in the biogenesis of the ER translocon (32Knight B.C. High S. Biochem. J. 1998; 331: 161-167Google Scholar), we chose Sec61β, one of the tail-anchored subunits of the Sec61 complex, for our initial studies. The β and γ subunits of the Sec61 complex both have amino acid sequences that are characteristic of tail-anchored proteins (30Hartmann E. Sommer T. Prehn S. Görlich D. Jentsch S. Rapoport T.A. Nature. 1994; 367: 654-657Google Scholar) and, like Syb2 (19Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Google Scholar), are capable of authentic post-translational membrane integration (data not shown). A preliminary time course experiment with Sec61β showed that during a 30-min incubation in the presence of canine pancreatic microsomes, more than 30% of the total protein synthesized was both membrane-associated and resistant to extraction with alkaline sodium carbonate solution, indicating that it was fully integrated (Fig.2 A; see also “Experimental Procedures” and Refs. 23Kutay U. Hartmann E. Rapoport T.A. Trends Cell Biol. 1993; 3: 72-75Google Scholar and 32Knight B.C. High S. Biochem. J. 1998; 331: 161-167Google Scholar). We then went on to compare samples of in vitro synthesized, membrane-integrated Sec61β incubated in the presence and absence of BMH and discovered a number of specific cross-linking products (Fig.2 B, lanes 1 and 2). In particular, prominent adducts of ∼22, ∼34, ∼40, and ∼50 kDa were seen (Fig.2 B, lane 2), suggesting the cross-linking of the ∼12-kDa Sec61β polypeptide chain to cellular components of ∼10, ∼22, ∼28, and ∼38 kDa, respectively. It should be noted that the apparent mobility of cross-linking products on SDS-PAGE might not accurately reflect the sum of the molecular masses of the cross-linked components. Hence, in the absence of additional information the predicted sizes of the cross-linking partners must be treated with caution. We analyzed the Sec61β cross-linking products by immunoprecipitation using a variety of antisera to ER components and were able to identify two of the major adducts unambiguously. Thus, newly synthesized membrane-integrated Sec61β was found to be cross-linked to the α subunit of the Sec61 complex (Fig. 2 B, lane 3) and SPC25, the 25-kDa subunit of the signal peptidase complex (Fig.2 B, lane 5). As expected, all of the adducts were immunoprecipitated by an antiserum recognizing Sec61β (Fig.2 B, lane 4). Furthermore, cross-linking of Sec61β to both Sec62p and Sec63p was also observed (Fig.2 B, lanes 6 and 7, respectively), although these did not appear to be major adducts (Fig. 2 B,lanes 2 and 4). Given that Sec61β forms part of the heterotrimeric Sec61 complex, we reasoned that newly synthesized, membrane-inserted Sec61β would probably display two types of associations with endogenous ER components. These would most likely be transient associations, indicative of a biosynthetic pathway, and stable associations, indicative of assembly into the Sec61 complex. We therefore carried out a time course experiment where membrane-associated Sec61β was isolated from the translation reaction and incubated for increasing periods of time before cross-linking was initiated. From this time course experiment, it was immediately obvious that all of the major cross-linking products except the Sec61α adduct reflected a transient association between the newly synthesized Sec61β polypeptides and ER-associated components (Fig. 2 C,lanes 1–5; cf. Fig. 2 B, lane 3). Thus, the pattern of BMH-dependent cross-linking products was the same as observed previously when cross-linking was carried out immediately after the isolation of the membrane fraction (Fig. 2 B, lane 2, and Fig. 2 C,lane 1). In contrast, when the membrane fraction was incubated for 10 min at the translation temperature before cross-linking, only the prominent Sec61α adduct remained (Fig.2 C, lane 2, product indicated by the filled circle in lane 6). The sample could be incubated at 30 °C for up to 120 min before adding the cross-linking reagent, and a strong adduct with Sec61α was still observed, whereas none of the other major products seen at the zero time point was detected (Fig.2 C, lanes 1–5). Interestingly, the “release” of newly synthesized Sec61β from its transient association with a discrete set of cellular components could be prevented by incubating the samples on ice rather than at 30 °C (Fig. 2 C, lanes 4 and 6). This observation indicates that the release of Sec61β from these transient associations is prevented at low temperature. We found that adduct formation with Sec61α was largely unaffected by prolonged incubation before initiating BMH-dependent cross-linking, confirming the stability of reduced thiol groups under the experimental conditions we have used. The prolonged association of Sec61β with Sec61α which we can detect by cross-linking presumably reflects the in vitro formation of Sec61-derived complexes. We did observe some reduction in the overall intensity of the Sec61β/Sec61α adduct across a 120-min time course, although the radiolabeled Sec61β chains were stable during this period (Fig.2 C, lanes 1–5). In contrast, the Sec61β/Sec61α adduct was unaffected by a 60-min incubation at 0 °C (Fig. 2 C, lanes 4 and 6). We believe that Sec61β can be cross-linked to Sec61α both during its biosynthesis, where the association is transient, and during subsequent complex formation, where the association is stable. In the case of complex formation, the radiolabeled Sec61β synthesized in vitro must compete for binding with endogenous unlabeled Sec61β present in the canine pancreatic microsomes. Such competition would limit the proportion of radiolabeled Sec61β that could be cross-linked to Sec61α, and presumably a “steady state” is reached. This is also consistent with our observation that between 30 and 120 min the efficiency of cross-linking to Sec61α is relatively constant (Fig. 2 C, lanes 3–5). On the basis of our cross-linking analysis with Sec61β, we concluded that BMH-dependent cross-linking could be exploited successfully to reveal transient associations between newly synthesized tail-anchored proteins and cellular components. To focus our analysis on potential biosynthetic interactions, we next analyzed a tail-anchored protein that would not be expected to form any stable complexes with endogenous ER components. For this reason we chose Syb2 as a previously well characterized tail-anchored protein that is membrane-integrated at the ER and then transported to post-ER vesicular structures (19Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Google Scholar). When wild type Syb2 was analyzed no BMH-dependent adducts were observed, indicating that the single naturally occurring cysteine residue present within the transmembrane domain of Syb2 (see Fig. 1) is insufficient to generate BMH-dependent adducts (data not shown). A number of previous studies have underlined the importance of the relative position of a cross-linking probe within a precursor upon its capacity for adduct formation (34Martoglio B. Dobberstein B. Trends Cell Biol. 1996; 6: 142-147Google Scholar). Given our success with the cross-linking analysis of Sec61β, we prepared a mutant form of Syb2 with a cysteine residue at an equivalent location within its cytosolic domain (Syb2cm, see Fig. 1). We confirmed that Syb2cm behaved as an authentic tail-anchored protein by showing that it could integrate post-translationally into canine pancreatic microsomes in the same way as the wild type protein (cf. Ref. 19Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Google Scholar and data not shown). As we had seen previously with Sec61β (Fig. 2, B andC), Syb2cm yielded several BMH-dependent cross-linking products when synthesized in the presence of ER-derived microsomal membranes (see Fig.3 A, lanes 1 and2). A parallel control experiment showed that no such BMH-dependent adducts were obtained with a version of Syb2cm which lacked its transmembrane domain (data not shown). All of the BMH-dependent adducts could be immunoprecipitated with an anti-Syb2 antibody confirming their origin (Fig. 3 A,lane 3). By screening a number of antisera recognizing known ER components, we were able to identify specific cross-linking of Syb2cm with Sec61β (∼10-kDa partner, Fig. 3 A, lane 5, *) and SPC25 (∼22-kDa partner, Fig. 3 A, lane 6, †). The ∼38-kDa product observed in the absence of BMH (Fig.3 A, lane 1, thin arrow) is distinct from the SPC25 adduct (Fig. 3 A, lane 6) and is most likely an SDS-resistant dimer of Syb2cm. A weaker ∼80 kDa adduct with Sec62p (Fig. 3 A, cf. lanes 7 and8) could also be observed after immunoprecipitation. All three of these components are known to be either a part of, or closely associated with, the Sec61 translocon. Despite this, we could detect no cross-linking of Syb2cm to Sec61α, the core subunit of the Sec61 translocon (Fig. 3 A, lane 4). This is in contrast to the behavior of newly synthesized Sec61β, which exhibited strong cross-linking to Sec61α which generated a discrete ∼50-kDa adduct (see Fig. 2 B). The identity of at least three other major Syb2cm cross-linking products remains to be determined (see “Discussion”). To assess how general the associations of newly synthesized Sec61β and Syb2cm with specific ER components were, we repeated a similar experiment using a third tail-anchored protein. Syn1A has only one cysteine present in its cytosolic domain, and this is located 121 residues from the presumptive transmembrane region (Fig. 1). Nevertheless, like the other two proteins analyzed, Syn1A also yielded multiple BMH- dependent cross-linking products (Fig.3 B, lane 1). As with Syb2cm, specific adducts of Syn1A with Sec61β (Fig. 3 B, lane 3, *), SPC25 (Fig. 3 B, lane 4, †), and Sec62p (Fig.3 B, lane 5, ∼90 kDa product) were identified. At least two other major adducts were detected (Fig.3 B, lane 1; Fig. 4,lane 7), but these could not be identified by immunoprecipitation (see “Discussion”). An additional product was brought down nonspecifically during the immunoisolation of the Sec61β, SPC25, and Sec62p adducts (Fig.3 B, lanes 3, 4 and 5,open arrowhead). However, in other experiments this product was not associated with these adducts after immunoprecipitation (cf. Fig. 4.). Once again, Syn1A showed no evidence of cross-linking to Sec61α (Fig. 3 B, lane 2). The authenticity of the Syn1A adducts with Sec61β, SPC25, and Sec62p was confirmed further by showing that no such products could be detected when control immunoprecipitations were carried out in the absence of BMH-dependent cross-linking (data not shown). On the basis of the data outlined above, we concluded that newly synthesized tail-anchored proteins associate with several generic ER components including the β subunit of the Sec61 complex and two Sec61 associated components (SPC25 and Sec62p). In contrast to many previous studies that had investigated cotranslationally inserted membrane proteins (see Ref. 2Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Google Scholar and references therein), no cross-linking of the tail-anchored" @default.
• W1988625335 created "2016-06-24" @default.
• W1988625335 creator A5005586711 @default.
• W1988625335 creator A5014887015 @default.
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• W1988625335 date "2003-02-01" @default.
• W1988625335 modified "2023-10-18" @default.
• W1988625335 title "Tail-anchored and Signal-anchored Proteins Utilize Overlapping Pathways during Membrane Insertion" @default.
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