Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition
2005; Springer Nature; Volume: 24; Issue: 14 Linguagem: Inglês
10.1038/sj.emboj.7600730
ISSN1460-2075
AutoresSilvia Brambillasca, Monica Yabal, Paolo Soffientini, Sandra Stefanovic, Marja Makarow, Ramanujan S. Hegde, Nica Borgese,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle23 June 2005free access Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition Silvia Brambillasca Silvia Brambillasca CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy Search for more papers by this author Monica Yabal Monica Yabal Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland Search for more papers by this author Paolo Soffientini Paolo Soffientini CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, ItalyPresent address: Istituto Nazionale per lo Studio e la Cura dei Tumori – Unità Trapianto Midollo 'Cristina Ghandini', 20133 Milano, Italy Search for more papers by this author Sandra Stefanovic Sandra Stefanovic Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Marja Makarow Marja Makarow Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland Search for more papers by this author Ramanujan S Hegde Corresponding Author Ramanujan S Hegde Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Nica Borgese Corresponding Author Nica Borgese CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy Faculty of Pharmacy, University of Catanzaro Magna Graecia, Roccelletta di Borgia (CZ), Italy Search for more papers by this author Silvia Brambillasca Silvia Brambillasca CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy Search for more papers by this author Monica Yabal Monica Yabal Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland Search for more papers by this author Paolo Soffientini Paolo Soffientini CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, ItalyPresent address: Istituto Nazionale per lo Studio e la Cura dei Tumori – Unità Trapianto Midollo 'Cristina Ghandini', 20133 Milano, Italy Search for more papers by this author Sandra Stefanovic Sandra Stefanovic Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Marja Makarow Marja Makarow Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland Search for more papers by this author Ramanujan S Hegde Corresponding Author Ramanujan S Hegde Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Nica Borgese Corresponding Author Nica Borgese CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy Faculty of Pharmacy, University of Catanzaro Magna Graecia, Roccelletta di Borgia (CZ), Italy Search for more papers by this author Author Information Silvia Brambillasca1, Monica Yabal2, Paolo Soffientini1, Sandra Stefanovic3, Marja Makarow2, Ramanujan S Hegde 3 and Nica Borgese 1,4 1CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy 2Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland 3Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 4Faculty of Pharmacy, University of Catanzaro Magna Graecia, Roccelletta di Borgia (CZ), Italy *Corresponding authors: Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, 18 Library Drive, Bldg. 18, Room 101, Bethesda, MD 20892, USA. Tel.: +1 301 496 4855; Fax: +1 301 402 0078; E-mail: [email protected] Institute of Neuroscience/Cell Mol Pharmacology, via Vanvitelli 32, 20129 Milano, Italy. Tel.: +39 02 503 16971; Fax: +39 02 749 0574; E-mail: [email protected] The EMBO Journal (2005)24:2533-2542https://doi.org/10.1038/sj.emboj.7600730 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition. Introduction In eukaryotic cells, the membranes that delimit organelles provide not only a compartment within which to sequester components, but also a membrane surface on which proteins with cytosolic activities can be spatially localized. These cytosolic activities are often carried by membrane proteins endowed with a single C-terminal hydrophobic domain capable of insertion into the lipid bilayer. Such tail-anchored (TA) proteins are found on all intracellular membranes exposed to the cytosol, and are involved in a remarkably diverse range of physiologic processes ranging from intracellular trafficking to protein degradation and programmed cell death (reviewed in Borgese et al, 2003). Thus, deciphering the molecular details underlying the selective membrane insertion and trafficking of TA proteins is critical to understanding a wide range of cell biological and physiological processes. Despite this importance, the mechanisms utilized by TA proteins to arrive at and insert into their target membrane are largely unresolved. The ER is the target of most newly synthesized TA proteins, since all those destined to compartments of the secretory pathway first insert into this membrane and then travel to their final residence by vesicular trafficking (Jäntti et al, 1994; Kutay et al, 1995; Linstedt et al, 1995; Pedrazzini et al, 1996). Insertion of membrane proteins into the ER has been studied most extensively for non-TA proteins, whose translocation and integration occurs by a ribosome-dependent cotranslational mechanism. A hydrophobic domain, either a signal sequence or a transmembrane segment, is recognized upon its emergence from the ribosome by the signal recognition particle (SRP). After targeting of this ribosome–nascent chain–SRP complex to the ER membrane, interactions between the Sec61 translocation channel and transmembrane segments mediate their subsequent cotranslational integration into the lipid bilayer (Rapoport et al, 1996). TA proteins stand apart from other membrane proteins, because their membrane anchor is still buried inside the ribosome when the stop codon is reached. Thus, it is clear that TA protein targeting and insertion into the lipid bilayer must occur by a ribosome-independent pathway after synthesis of the entire protein has been completed. What remains unknown is whether qualitatively distinct components and mechanisms are involved in the insertion of TA and non-TA membrane proteins into the lipid bilayer. Studies attempting to address this issue have yielded differing results. For one commonly used model TA protein, cytochrome b5 (b5), functional analyses using yeast mutants defective in the Sec61 system showed no impairment of b5 insertion (Yabal et al, 2003), arguing that the phenomenon occurs independently of the known Sec61 functions. However, these results did not exclude the possibility that the Sec61 translocon does function in b5 insertion, but by a mechanism not affected by the analysed mutations. For another widely studied TA protein, synaptobrevin, some investigators found that its insertion in vitro can proceed independently of either the Sec61 complex or SRP receptor (Kutay et al, 1995), although at least one ER membrane protein has been implicated (Kutay et al, 1995; Kim et al, 1997). On the other hand, cross-linking experiments revealed a transient association of the in vitro synthesized protein with Sec61 (Abell et al, 2003), as well as an unexpected post-translational interaction with SRP54 (Abell et al, 2004). While a functional role for the Sec61 complex remains to be established, synaptobrevin binding to membranes in vitro was decreased in the absence of either SRP or its receptor (Abell et al, 2004). One problem in interpreting in vitro studies on TA protein insertion is that most of the reported results are based on membrane-binding assays that may not necessarily measure correct transmembrane integration. This problem is well exemplified by the studies with b5. There has been general agreement that both the purified protein isolated from tissues and the in vitro synthesized polypeptide are capable of binding to a wide range of natural and artificial membranes, including protease-treated microsomes and protein-free liposomes (reviewed in Borgese et al, 2003). However, whereas the native protein in vivo is inserted in the ER with transmembrane topology (Kuroda et al, 1996; Pedrazzini et al, 2000), the purified protein can associate tightly with lipid bilayers in a hairpin conformation, that is, with the N and C termini both exposed to the outside of the vesicles (Dailey and Strittmatter, 1981; Arinc et al, 1987). The capability of b5 to bind to lipid bilayers with an unphysiological topology may explain the discrepancy between its specific ER localization in vivo (D'Arrigo et al, 1993) and its promiscuity in in vitro assays (Remacle, 1978; Kim et al, 1997). To overcome the difficulties inherent in binding assays, we previously used an epitope-tagged b5 containing an N-glycosylation site close to the C-terminus. By adopting glycosylation as a stringent criterion for ER translocation, a transmembrane topology for b5 could be demonstrated in vivo and in vitro (Pedrazzini et al, 2000; Borgese et al, 2001; Yabal et al, 2003). One drawback of this assay is that it requires all of the components for the N-glycosylation reaction to be present and active in the membrane. For this reason, the glycosylation assay is not useful for in vitro work with proteoliposomes reconstituted from fractionated microsomal extracts, an approach that has been of fundamental importance for the identification of membrane components involved in cotranslational translocation (Nicchitta et al, 1991; Görlich and Rapoport, 1993). The application of this same approach to the problem of TA protein insertion therefore necessitates an appropriately specific and quantitative translocation assay. Here, we have developed a rigorous assay for transmembrane integration of a TA protein based on protection from proteolysis of the translocated C-terminal tail. This assay allowed us to quantitatively assess both the efficiency and kinetics of topologically correct b5 insertion into proteoliposomes reconstituted from systematically fractionated ER microsomal components. The results from this analysis, together with the demonstration of efficient transmembrane insertion into protein-free liposomes, argue strongly against either essential or even stimulatory membrane protein requirements. Instead, insertion efficiency is strikingly modulated by alterations in lipid composition of the vesicles, a finding that may explain the long-observed in vivo targeting specificity of b5, as well as of other ER-directed TA proteins. Results Assay design and characterization We devised and tested a protease protection approach to selectively and directly detect the properly inserted C-terminal tail of a model TA protein (Figure 1A). The substrate we chose is a b5 variant, called b5-Nglyc, that has been well characterized in vitro and in vivo (Pedrazzini et al, 2000; Yabal et al, 2003). The C-terminus of this substrate contains not only a glycosylation site that facilitates optimization and validation of the protease assay, but also a 19-residue sequence (from the N-terminus of bovine opsin) that is recognized by a monoclonal antibody (Adamus et al, 1991) (see Figure 1A). In this assay, post-translational translocation of the C-terminus of b5-Nglyc across the membrane of a closed vesicle should result in protease protection of a ∼5 kDa peptide that contains three of five 35S-methionine residues, an intact opsin epitope, and, depending on the membranes used, a single high-mannose carbohydrate tree (which would increase the apparent size by ∼4 kDa). While glycosylation serves as an independent marker of translocation, it is important that it not be required if the assay is to be useful in fractionated proteoliposomes. Hence, immunological detection of the protected C-terminal peptide should serve as a specific and quantitative marker of proper b5-Nglyc topogenesis. Figure 1.Protease protection assay for transmembrane insertion of a TA protein. (A) Illustration of the model TA protein construct (b5-Nglyc) and assay design. (B) In vitro translated b5-Nglyc was post-translationally incubated with ER-derived RMs in the absence or presence of a tripeptide inhibitor of glycosylation (NYT). The samples were then divided for digestion with PK in the presence or absence of the detergent TX100. Equal amounts of each sample were subsequently immunoprecipitated with antibodies against the C-terminal opsin tag prior to analysis by SDS–PAGE and autoradiography. The positions of the primary translation product (b5), glycosylated b5 (g-b5), and their respective protease-protected fragments (PF and g-PF) are indicated to the left. The efficiency of insertion (% ins) is indicated below the respective lanes. (C) Time course of glycosylation and translocation of b5-Nglyc. Translated b5-Nglyc was incubated with RMs for the indicated times. For each time point, total nondigested and PK-digested immunoprecipitated samples are shown in the upper and lower panel, respectively. A nonglycosylated PF band could not be detected even on longer exposures of the autoradiograph. (D) Time course of generation of b5-Nglyc PF with different loads of pig RM: 0.1 eq/μl (•); 0.35 eq/μl (▪); 0.7 eq/μl (▴). In all figures, numbers on the side of the panels indicate the position and size (in kDa) of Mr markers. Download figure Download PowerPoint These expectations were borne out by the experiment illustrated in Figure 1B. Approximately 50% of the ∼20 kDa b5-Nglyc polypeptide (indicated as b5 in lane 1) generated by in vitro translation in reticulocyte lysate (RL) was converted by post-translational incubation with ER-derived rough microsomes (RMs) into a glycosylated form (g-b5, lane 3). When the translation mix without added RM was exposed to proteinase K (PK), no immunoprecipitable b5-Nglyc fragments were detected (lane 2), even after gross overexposure of the autoradiograph (not shown). By contrast, post-translational incubation with RM prior to PK digestion generated an ∼9 kDa protected fragment (g-PF, lane 4) that was immunoprecipitated by the opsin antibody. The g-PF band was not seen if detergent was included during the PK digestion, demonstrating that its generation is dependent on an intact membrane (lane 5). The identification of g-b5 and g-PF as glycosylated products was verified by their markedly reduced generation when parallel reactions were performed in the presence of a tripeptide (NYT) that competitively inhibits glycosylation (lanes 6–8). In this case, PK treatment generated a ∼5.5 kDa PF (lane 7), which, like g-PF, was digested in the presence of detergent (lane 8). These results not only demonstrated the high sensitivity and specificity of the assay, but also indicated that glycosylation itself is not required to stabilize the transmembrane topology of b5-Nglyc (lane 7 versus 4). In addition, analysis of the nonimmunoprecipitated products (Supplementary Figure S1) demonstrated the absence of topologically incorrect b5-Nglyc transmembrane integration. Since PK (at the 0.25 mg/ml concentration we routinely used) can substantially digest substrates within seconds after addition (data not shown), we reasoned that the protection assay could also be used to monitor the kinetics of b5-Nglyc insertion. A comparison of the time course of glycosylation and of PF generation (Figure 1C) showed that the two phenomena proceeded in parallel, since no unglycosylated peptide was detected even at early time points. These results demonstrate that the rate-limiting step for glycosylation in RM is translocation across the microsomal membrane. Furthermore, insertion under these conditions progresses steadily for over 30 min, achieving the maximal translocation of ∼50% only after ∼45 min. This moderate efficiency and kinetics allows both easily detectable translocation and any potential changes that may be caused by manipulations to membrane composition. Indeed, changing the amount of RM in the reaction results in readily detectable changes in transmembrane insertion of b5-Nglyc, with increasing rate and efficiency at higher concentrations of vesicles (Figure 1D). Since glycosylation in RM proceeds essentially simultaneously with insertion, glycosylation efficiency (which is readily evaluated in the undigested sample) provides a direct measure of insertion efficiency. Accordingly, the immunoprecipitated g-PF band for the RM sample represents a quantitative standard for this level of insertion. This means that the g-PF band can be compared to the PF and/or g-PF bands of parallel samples to judge their insertion efficiencies relative to this standard. Thus, as long as an insertion reaction with RM is analysed in parallel, b5-Nglyc insertion can be readily quantified even in the absence of glycosylation (e.g., in reconstituted proteoliposomes, as shown in Figures 2 and 3, and Supplementary Figure S5). Figure 2.Transmembrane integration of b5-Nglyc is unaffected by Sec61 depletion. (A) Proteoliposomes reconstituted from a Sec61-depleted extract or a depleted extract replenished with purified Sec61 complex were analysed by immunoblotting against various translocation-related ER proteins (SRα, α subunit of the SRP receptor; RbI, ribophorin I; TRAM, translocating chain-association membrane protein; TRAPα, α subunit of the translocon-associated protein; SPC, signal peptidase complex). (B) The Sec61-depleted and replenished proteoliposomes from panel A were assayed for cotranslational translocation of pPL. Equal aliquots of the translocation reaction were either left untreated (top panel) or digested with PK (bottom panel) prior to analysis by SDS–PAGE. Translocation reactions containing native RM, proteoliposomes made with a total RM extract (Total), or without added vesicles (Φ) are also included for comparison. The positions of precursor (pPL) and signal-cleaved PL are indicated. (C) The various membranes analysed in panel B were assayed for b5-Nglyc translocation using the protease protection assay. (D) The time course of b5-Nglyc translocation into Sec61-depleted and replenished proteoliposomes was assayed by protease protection. Reactions lacking vesicles (Φ) or containing RM are also shown. Download figure Download PowerPoint Figure 3.Proteoliposomes made with differently depleted extracts support b5-Nglyc transmembrane insertion with equal efficiency. (A) Immunoblot analysis of proteoliposomes reconstituted from either a total RM extract, ConA-depleted extract, Q-Sepharose-depleted extract, or the depleted extracts replenished with the material eluted from the corresponding resins (+Elu.). Abbreviations are as in the legend to Figure 2. The various proteoliposomes from panel A were analysed for post-translational translocation of b5-Nglyc (B) or cotranslational translocation of pPL (C) and PrP (D). For pPL and PrP, only the PK digested samples are shown. The PF band indicative of b5-Nglyc translocation into proteoliposomes was not observed if detergent was included during the protease digestion reaction (Supplementary Figure S4). The positions of the fully translocated form of PrP (termed secPrP) and the protected fragments generated from two transmembrane forms (termed CtmPrP and NtmPrP) are indicated by 'sec', 'Ctm', and 'Ntm', respectively (Fons et al, 2003). Download figure Download PowerPoint Analysis of b5 translocation upon Sec61 depletion To examine the membrane requirements for b5 translocation, it is necessary to reassemble translocation-competent proteoliposomes using components solubilized from RM. Using conditions that had previously been used to dissect the requirements for cotranslational translocation, we first examined the role of the Sec61 complex, the only known protein-conducting channel of the mammalian ER (Rapoport et al, 1996). A microsomal detergent extract was immunodepleted of the Sec61 complex and then reconstituted into proteoliposomes either before or after replenishment with purified Sec61 complex to the original levels. By immunoblot analysis, both Sec61α and Sec61β were undetectable in the immunodepleted proteoliposomes, while other RM components were unaffected (Figure 2A and Supplementary Figure S2). As shown in Figure 2B, translocation of the cotranslational signal peptide-containing substrate preprolactin (pPL) into the Sec61-depleted proteoliposomes was sharply impaired, as judged by a lack of both signal peptide cleavage and protease protection. Replenishment with Sec61 allowed pPL translocation to levels comparable to unfractionated proteoliposomes (which are still much less efficient than native RMs). In sharp contrast to pPL, translocation of the C-terminus of b5-Nglyc proceeded equally well across Sec61-depleted, replenished, or unfractionated proteoliposomes (Figure 2C), as revealed by generation of the ∼5.5 kDa PF. Although reconstituted proteoliposomes are rather inefficient in N-glycosylation (Görlich et al, 1992), longer exposures revealed a small but equal degree of glycosylation of b5-Nglyc in the depleted or replenished proteoliposomes (and corresponding g-PF after PK digestion). This independently confirmed a lack of effect of Sec61 depletion on b5-Nglyc transmembrane integration. We also evaluated the possibility that slow but eventual complete insertion of b5-Nglyc was responsible for the lack of effect after the 1 h translocation reaction in Figure 2C. However, a time course of b5-Nglyc translocation into the depleted and replenished proteoliposomes revealed comparable efficiencies and rates of insertion in both cases (Figure 2D). Of note, Figure 2C and D revealed that the overall efficiency of b5-Nglyc translocation into the proteoliposomes was even higher than that seen with native RM. This is markedly different from cotranslational translocation (Figure 2B), which is far less efficient in the proteoliposomes than in RM, despite the two preparations containing comparable amounts of various translocon components (Supplementary Figure S2). This suggested that features of the membrane required for TA protein insertion are functionally reconstituted far more efficiently than the components needed for cotranslational translocation. This observation is discussed further below, after presentation of the data of Figure 4. Figure 4.Post-translational translocation of b5-Nglyc into protein-free liposomes. (A) Standard b5-Nglyc translocation reactions were performed for 90 min with protein-free liposomes prepared from PC, a mixture of PC and PE in a 4:1 ratio, or total lipids extracted from RM (RM lipids). Liposomes were made either using extrusion of a detergent-free lipid suspension (lanes 3–5) or by removal of detergent (DBC method, lanes 6 and 7). Reactions in the absence of added vesicles (Φ) or with pig RMs in the presence of NYT were analysed in parallel. A total of 10 μg phospholipid per 10 μl reaction volume was used in lanes 2–7. The PF band indicative of b5-Nglyc translocation into liposomes was not observed if detergent was included during the protease digestion reaction (Supplementary Figure S4). (B) Time course of b5-Nglyc translocation into protein-free liposomes (prepared by extrusion) compared with pig RMs. The amount of PF generated by the standard protease protection assay was quantified by phosphorimaging and plotted on the y-axis. For the RM sample, the sum of PF and g-PF, the latter due to incomplete inhibition of glycosylation by the NYT tripeptide, is given. Download figure Download PowerPoint Evaluation of membrane protein requirements during b5 translocation Having excluded the involvement of the Sec61 complex, we next turned to other microsomal proteins that might facilitate b5-Nglyc insertion. We first fractionated microsomal detergent extracts by chromatographic procedures based either on anion exchange (Q-sepharose) or on affinity depletion of glycoproteins by immobilized concanavalin A (ConA), and used the depleted extracts to reconstitute proteoliposomes. Western blot analysis of the proteoliposomes demonstrated that numerous proteins known or implicated in translocation were depleted in at least one of the samples by more than 90% (Figure 3A and Supplementary Figure S3). However, we could not detect any difference in b5-Nglyc translocation into the fractionated proteoliposomes compared to the unfractionated controls (Figure 3B, lanes 4 and 6 versus lane 3), and replenishment of the depleted proteoliposomes with material eluted from the respective resins had no effect on the extent of translocation (lanes 5 and 7). Insertion efficiencies into the proteoliposomes (more than 90% in each case) were again better than into RM (∼60%). Notably, the ribophorin I depletion mediated by ConA (panel A) did impair glycosylation of b5-Nglyc (lane 4), as expected. The functional effectiveness of the fractionation and reconstitution procedures was further verified by analyses of the classical pPL substrate and of Prion protein (PrP) translocation. The latter differs from pPL in its requirement for the TRAP complex for complete translocation into the ER lumen, in the secPrP form (Fons et al, 2003). The absence in ConA-depleted proteoliposomes of the signal peptidase complex (Figure 3A) was functionally correlated to lack of pPL signal cleavage, but not its translocation (Figure 3C, lane 4). Similarly, complete translocation of PrP across the membrane (secPrP form, indicated as sec in Figure 3D) was inhibited in both the ConA- and Q-depleted proteoliposomes (Figure 3D, lanes 4 and 6), both of which lack the TRAP complex (Figure 3A). Thus, while the fractionation procedures clearly influenced various translocon-related activities, the proper transmembrane insertion of b5-Nglyc was unchanged. Proteoliposomes made from numerous other ion-exchange fractions or protease-digested detergent extracts also supported efficient transmembrane insertion of b5-Nglyc (Supplementary Figure S5). Taken together, these results suggested that no single microsomal protein or lipid species was specifically required for b5-Nglyc translocation. Furthermore, it appears that bulk proteins do not stimulate or inhibit b5-Nglyc insertion nonspecifically. This conclusion is based on the collective observations in Figures 2 and 3, and Supplementary Figure S5 that protein mixtures of vastly different composition and abundance have little influence on b5-Nglyc insertion when incorporated into lipid vesicles. Analysis of translocation across protein-free liposomes The above results together suggested that the C-terminus of in vitro synthesized b5-Nglyc can be translocated completely across the lipid bilayer without the aid of any proteins. To test this hypothesis, we analysed b5-Nglyc translocation into protein-free liposomes of differing composition. Phosphatidylcholine (PC), a mixture of PC and phosphatidylethanolamine (PE) in the ratio approximating that in ER membranes (Colbeau et al, 1971), or total lipids extracted from RM, were used to prepare the liposomes using either the detergent removal method or a detergent-free extrusion procedure (see Materials and methods). All preparations were present at the same phospholipid concentration (1 μg phospholipids/μl) so as to provide comparable membrane surface areas. As shown in Figure 4, with all liposome preparations, we obtained translocation efficienc
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