Role of calnexin in the glycan-independent quality control of proteolipid protein
2003; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês
10.1093/emboj/cdg300
ISSN1460-2075
Autores Tópico(s)RNA regulation and disease
ResumoArticle16 June 2003free access Role of calnexin in the glycan-independent quality control of proteolipid protein Eileithyia Swanton Corresponding Author Eileithyia Swanton School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Stephen High Stephen High School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Philip Woodman Philip Woodman School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Eileithyia Swanton Corresponding Author Eileithyia Swanton School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Stephen High Stephen High School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Philip Woodman Philip Woodman School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Author Information Eileithyia Swanton 1, Stephen High1 and Philip Woodman1 1School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK ‡S.High and P.Woodman contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2948-2958https://doi.org/10.1093/emboj/cdg300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins. Introduction The endoplasmic reticulum (ER) is a major site for the synthesis of membrane and secretory proteins. The lumen of the ER contains a variety of molecular chaperones and folding factors which assist the folding and oligomeric assembly of newly synthesized proteins. These include the Hsp 70 family member BiP, the ER-resident lectins calnexin and calreticulin, and the oxidoreductases PDI and ERp 57. In order to restrict the deployment of potentially toxic misfolded or unassembled proteins within the cell, the ER has developed stringent quality control systems to ensure that only correctly folded and assembled proteins are allowed to exit and proceed along the secretory pathway (Hurtley and Helenius, 1989). Most proteins that fail this quality control system are ultimately degraded by the cytosolic proteasome through a process termed ER-associated degradation (ERAD) (Wiertz et al., 1996). A common post-translational modification that occurs in the ER is the addition of N-linked glycans, and the mechanisms of glycoprotein-specific quality control have been particularly well studied (Ellgaard and Helenius, 2001). Trimming of the triply glucosylated glycan that is added to the growing polypeptide produces a monoglucosylated form which allows the glycoprotein to interact with calnexin and calreticulin in combination with ERp 57 (High et al., 2000). Removal of the final glucose from the N-linked glycan releases the glycoprotein from calnexin/calreticulin, and if the glycoprotein has attained its native conformation it will exit the ER. If not, reglucosylation of the glycan by a glucosyl transferase that specifically recognizes misfolded proteins causes the glycoprotein to rebind calnexin/calreticulin, thereby retaining it in the ER and providing further time for folding. Terminally misfolded glycoproteins are released from this cycle of calnexin/calreticulin binding by trimming of mannose residues in the glycan. This inhibits reglucosylation, and thus the interaction with calnexin/calreticulin, and targets the protein for degradation (Braakman, 2001; Cabral et al., 2001). In contrast, very little is know about the quality control of non-glycosylated proteins, although a system is presumed to exist. The growing list of diseases that are associated with the ER retention of membrane and secretory proteins (not all of which are glycosylated) highlights the importance of understanding the molecular mechanisms that underlie the quality control of non-glycosylated as well as glycosylated proteins (Brooks, 1997). We have used proteolipid protein (PLP) as a model for non-glycosylated polytopic membrane proteins to examine the quality control systems that exist for this class of polypeptides. PLP is expressed primarily by oligodendrocytes and Schwann cells, and is the major membrane protein of central nervous system myelin. The structure of PLP is relatively simple compared with other polytopic proteins; it has four transmembrane (TM) domains, a large extracellular/ER lumenal domain between The third and fourth TM domains, and short cytosolic N- and C-terminal domains (Figure 1A). PLP has two disulfide bonds within its large extracellular domain and up to six sites for palmitoylation. PLP interacts with lipid rafts during its transit from the ER to the cell surface (Simons et al., 2000), where it is incorporated into the myelin membrane. Two features make PLP a particularly useful model for the study of ER protein quality control. First, it has no consensus sites for N-glycosylation and therefore will interact with components of the putative glycan-independent quality control system. Secondly, a large number of naturally occurring point mutations in the sequence of PLP are associated with diseases of the central nervous system, including Pelizaeus–Merzbacher disease. Most of these mutations prevent PLP from reaching the cell surface, and the mutant protein appears to be misfolded and retained in the ER (Gow and Lazzarini, 1996), indicating that it is recognized by an ER quality control system. Figure 1.Structure and subcellular distribution of wt and msd PLP. (A) Cartoon depicting the topology of PLP. Palmitoylation sites are shown by squiggles, disulfide bonds are shown by lines, the position of the msd mutation in TM4 is shown by an asterisk and the position of the artificial glycosylation sites (CHO1 and CHO2) are shown by arrowheads. (B) COS-7 cells were transiently transfected with wt (left and center column) or msd (right column) PLPmh, fixed with 2% formaldehyde and 0.2% gluteraldehyde, permeabilized and stained for myc (top row; green), PMCA, Lamp 2 or calnexin (cnx) (center row, red). Merged images are shown in the bottom row. Bar, 20 μm. (C) COS-7 cells were transfected with PLPha (lanes 1–2, 7–8), PLPha containing artificial glycosylation site CHO1 (lanes 3–4, 9–10) or CHO2 (lanes 5–6, 11–12), labeled with [35S]methionine/cysteine for 2 h, solubilized and immunoprecipitated with anti-ha and and left untreated (−) or treated with EndoH (+). PLPha (− CHO) and artificially glycosylated PLPha (+ CHO) are shown by arrows. Download figure Download PowerPoint Here, we have used a well-characterized folding mutant of PLP to study the glycan-independent quality control pathways operating in the ER. We find that calnexin binds to newly synthesized PLP, and show that it contributes directly to the ER quality control of PLP. Notably, calnexin interacts with the fourth TM domain of PLP, binding stably to it when it is misfolded or unassembled. We provide evidence that this interaction with calnexin inhibits the degradation of misfolded PLP, thereby exacerbating the potentially toxic accumulation of an ER-retained protein. Results Subcellular localization of wild-type and misfolded PLP In order to facilitate subsequent analysis, we added either a myc/His6 (mh) or ha epitope tag to the C-terminus of PLP (Figure 1A). This region of PLP is the most variable between species and contains few disease-associated mutations, suggesting that by locating tags here we were unlikely to interfere with correct folding of the protein. A well studied mutation, the msd allele, that causes complete ER retention of the mutant protein and results in severe disease in transgenic mice is the substitution of a valine for an alanine at position 242 in the fourth TM domain of PLP (Figure 1A, asterisk). We have used this as a natural folding mutant with which to examine the quality control of a non-glycosylated membrane protein. The subcellular distribution of wt and msd PLP in COS-7 cells was examined by indirect immunofluorescence microscopy (Figure 1B). The majority of wt PLPmh was transported to the cell surface and colocalized with the plasma membrane ATPase PMCA, with a variable amount seen in intracellular structures that colocalized with the lysosomal marker Lamp 2, as has been seen previously in cultured cells (Gow and Lazzarini, 1996; Simons et al., 2002). In contrast, staining of msd PLPmh was restricted to a reticular pattern typical of the ER (Figure 1B). This was confirmed by costaining with the ER marker calnexin, which colocalized with the msd form of PLPmh. Although the misfolded msd PLP appeared to concentrate in a perinuclear region, we found no evidence of aggresome formation even in the presence of proteasome inhibitors, and no rearrangement of vimentin filaments characteristic of aggresomes (Johnston et al., 1998) occurred under these conditions (data not shown). Similar results were obtained with untagged wt and msd PLP and wt and msd PLPha (data not shown). These results demonstrate that the intracellular trafficking of the epitope-tagged PLPs is similar to that of the untagged proteins in COS-7 cells. More strikingly, it is clear that the msd form of PLP is recognized as misfolded and retained in the ER by quality control machinery that acts independently of N-glycosylation. In order to demonstrate that epitope-tagged PLP has the orientation shown in Figure 1A, we introduced artificial glycosylation sites into the first (CHO1, Asn 47) and second (CHO2, Asn 222) lumenal loops of wt and msd PLPha. As shown in Figure 1C, both sites were glycosylated, resulting in an Endo H-sensitive shift in the molecular weight of the protein. This confirms that these regions are located within the ER lumen and that the msd mutation does not alter the predicted orientation of the protein, as previously shown by Gow et al. (1997). Similar results were obtained when the protein was synthesized in a cell-free system supplemented with semipermeabilized mammalian cells (Figure 2A), confirming that the topology of PLP is maintained in this in vitro system. Figure 2.Interaction of newly synthesized PLP with glycan-independent chaperones. (A) [35S]PLPha (lanes 1–2, 7–8), PLPha containing artificial glycosylation site CHO1 (lanes 3–4, 9–10) or CHO2 (lanes 5–6, 11–12) was translated in vitro for 1 h, immunoprecipitated with anti-ha and left untreated (−) or treated with EndoH (+). PLPha (− CHO) and artificially glycosylated PLPha (+ CHO) are shown by arrows. (B) 35S-labeled wt PLP was translated in vitro for 15 min, incubated at 30°C for 45 min and co-immunoprecipitated with anti-chaperone or control (ctl) antibodies as indicated. Lane 1 shows 25% of the total amount of PLP used for each immunoprecipitation, and 5 mM ATP was added to the immunoprecipitation buffer where indicated. (C) 35S-labeled wt or msd PLP was translated in vitro for 15 min and incubated at 30°C for the time indicated. Then, the samples were split in two, depleted of ATP and immunoprecipitated with anti-BiP or anti-calnexin (Figure 4B). Co-immunoprecipitated PLP (top panel) was quantified and expressed as a percentage of the total input. The graph (bottom panel) shows the mean ± SEM of three independent experiments. Download figure Download PowerPoint In vitro analysis of PLP folding; the role of glycan-independent chaperones In order to establish whether any previously identified 'glycan-independent' chaperones are involved in the folding of PLP, we synthesized the polypeptide in a cell-free system supplemented with semipermeabilized mammalian cells and co-immunoprecipitated the radiolabeled PLP with various anti-chaperone antibodies (Figure 2B). Some newly synthesized PLP was co-immunoprecipitated with cytosolic Hsc 70 and its cofactor Hsp 40, and a smaller amount of PLP co-immunoprecipitated with PDI. The strongest association that we could detect was with BiP (Figure 2B, lane 4), a well characterized chaperone that is regulated by ATP hydrolysis (Munro and Pelham, 1986). The authenticity of the interaction between newly synthesized PLP and BiP was confirmed by showing that the majority of PLP could be released by the addition of ATP (Figure 2B, compare lanes 4 and 5), and by mixing experiments which show that BiP does not bind to PLP following cell lysis (see Supplementary figure 2 available at The EMBO Journal Online). A strong interaction of PLP with BiP is consistent with studies showing that nascent proteins preferentially bind to calnexin and calreticulin if glycosylated within 50 residues of the N-terminus, but are likely to associate with BiP in the absence of glycosylation (Molinari and Helenius, 2000). We next examined the kinetics of BiP binding to newly synthesized wt and msd PLP in vitro by co-immunoprecipitation of radiolabeled PLP with anti-BiP at different times following the termination of translation (Figure 2C). A transient interaction of the wt protein with BiP was observed, which peaked at 30–60 min and decreased to background levels by 4 h after synthesis. In contrast, BiP bound stably to msd PLP over the time course of the experiment. These results show that BiP is able to distinguish between the wt and misfolded msd PLP, forming a stable complex with the misfolded protein. Such a complex would be retained in the ER by virtue of BiP's KDEL retrieval motif, and therefore could play a key role in preventing misfolded PLP leaving the ER. In vivo analysis In order to examine the role of BiP in the quality control of misfolded PLP in vivo, we generated stable HeLa cell lines expressing wt or msd PLPmh under the control of a tetracyclin inducible promoter. The distribution of wt and msd PLPmh in these cell lines (Figure 3A) was essentially the same as seen in transiently transfected COS-7 cells (cf. Figure 1B). Western blotting of cell lysates confirmed that, following induction with deoxycyclin, both cell lines synthesized a myc-tagged protein with a molecular weight corresponding to that of PLP (Figure 3B, arrowhead). A higher molecular weight myc-reactive product was also induced in cells expressing msd PLPmh (Figure 3B, asterisk). This form is also observed when msd PLPmh or ha are expressed transiently and most likely represents an SDS-resistant dimer of msd PLP. Figure 3.In vivo analysis of PLP folding. (A) Cell lines expressing wt (left) or msd (right) PLPmh were induced overnight, fixed with methanol and stained with anti-myc followed by FITC-conjugated secondary antibody. (B) Lysates of cells induced to express wt or msd PLP for the time indicated were western blotted with anti-myc followed by an HRP-conjugated secondary antibody. PLPmh is shown by an arrowhead and a putative PLP dimer by an asterisk. The anti-myc cross-reactive band just above the asterisk is present in all samples even without induction. (C) Stable cell lines were induced to express wt or msd PLPmh overnight, solubilized and immunoprecipitated with anti-myc or control (ctl) antibody. Co-immunoprecipitated material was western blotted with anti-BiP. Immunoprecipitates from uninduced msd PLPmh cells (msd unind), and from cells solubilized with buffer containing 5mM ATP are shown where indicated. An eighth of the total lysate used for immunoprecipitation is shown in lane 9. The heavy chain of the immunoprecipitation antibody is just visible at the bottom of the panel. (D) An eighth of the total lysates used for immunoprecipitation in (C) were western blotted with anti-myc. Each lane was exposed to film for the same time to allow comparison of the amount of PLPmh in each sample. (E) COS-7 cells were transfected with BiPSmyc or BiPKDELmyc. After 24 h, the cells and medium were separated and immunoprecipitated with mouse anti-myc; the immunoprecipitated material was western blotted with rabbit anti-myc. (F) COS-7 cells were cotransfected with BiPSmyc and msd PLPha, fixed with methanol and stained with anti-myc (left) and anti-ha (right) followed by FITC or Texas Red conjugated secondary antibodies, respectively. Images also show nuclei stained with DAPI. Bar, 20 μm. Download figure Download PowerPoint When PLP was immuno-isolated via its myc tag, we found that BiP could be co-immunoprecipitated with msd but not wt PLPmh (Figure 3C, compare lanes 1 and 5), demonstrating that BiP binds stably to misfolded PLP in vivo. Inclusion of ATP in the lysis buffer abolished co-immunoprecipitation of BiP with msd PLPmh, and no co-immunoprecipitation was seen in the absence of induction or with a control antibody (Figure 3C, lanes 3 and 6). Very little BiP co-immunoprecipitated with wt PLPmh in vivo, reflecting the transient nature of the chaperone–wt protein interaction. The failure to co-immunoprecipitate BiP with wt PLPmh was not due to lower levels of PLP expression, since similar levels of wt and msd PLPmh were found in the total cell lysates (Figure 3D). BiP is not solely responsible for the ER retention of misfolded PLP We reasoned that if the retention of misfolded PLP is mediated primarily through binding to BiP, overexpression of a secreted form of BiP in which the KDEL sequence was replaced with an myc tag (hereafter referred to as BiPS) would be expected to cause at least some msd PLP to leave the ER and move with BiPS to the cell surface. A similar approach has been used to demonstrate that PDI is responsible for the retention of unassembled procollagen C-propeptides (Bottomley et al., 2001). Although the ratio of BiPS to endogenous BiP was approximately 10:1 (Supplementary figure 3) and the majority of BiPS exits the ER and is secreted into the medium (Figure 3E), we saw no staining of msd PLPha at the cell surface or in Lamp 2-positive structures in cells that co-expressed BiPS (Figure 3F). This indicated that the misfolded PLPha was unable to leave the ER when co-expressed with BiPS. Hence, although misfolded PLP interacts strongly with BiP, this cannot be the only mechanism by which it is retained in the ER and additional pathways must exist for the quality control of non-glycosylated membrane proteins. Calnexin, but not calreticulin, binds misfolded PLP In order to identify other ER factors that might contribute to the quality control of msd PLP, we immunoprecipitated wt and msd PLPmh from induced cells under non-denaturing conditions and screened the products with a panel of antibodies to known ER chaperones. Given that PLP lacks N-linked gycans, we were surprised to observe a particularly strong association of msd, but not wt PLPmh, with the ER lectin calnexin (Figure 4A, lanes 1 and 3). We examined the kinetics of calnexin binding to newly synthesized PLP in vitro as described above for BiP. As shown in Figure 4B, calnexin interacts transiently with wt PLP, but binds stably to the misfolded protein. These results demonstrate that calnexin, like BiP, is able to distinguish between wt and misfolded msd PLP, forming a stable complex with the misfolded protein. Since calnexin can bind to wt PLP, we established whether the stable association with calnexin we detected in vivo was specific for misfolded PLP or whether it was due to the ER localization of msd but not wt PLPmh. To do this, we artificially retained wt PLPmh in the ER by adding Brefeldin A (BFA) to cells when they were induced with deoxycyclin. Whilst this treatment caused all the wt PLPmh to be retained in the ER (data not shown), it had no significant effect upon the amount of wt PLPmh that we found associated with calnexin (Figure 4C, compare lanes 2 and 4). In contrast, a significant amount of calnexin was bound to the msd PLPmh in both the absence and presence of BFA (Figure 4C, compare lanes 6 and 8). Hence, we conclude that, like BiP, calnexin is able to monitor the folding of a non-glycosylated membrane protein and bind stably to a misfolded version. The association of msd PLPmh with calnexin was not inhibited by prolonged treatment of the cells with tunicamycin or castanospermine (Figure 4D, lanes 1–6), both of which considerably reduce the glycan-mediated association of calnexin with a control glycoprotein opsin (Figure 4D, lanes 10–15). These results confirm that the interaction between calnexin and PLP is not dependent on glycosylation or glucose trimming, and rule out the unlikely possibility that calnexin binding to PLP occurs via a nascent intermediate factor that is itself N-glycosylated. Figure 4.Calnexin binds stably to misfolded PLP. (A) Stable cell lines were induced to express wt or msd PLPmh overnight, solubilized and immunoprecipitated with anti-myc or control (ctl) antibodies. Co- immunoprecipitated material was western blotted with anti-calnexin (cnx). An eighth of the total lysates used for immunoprecipitation is shown in lanes 5 and 6. (B) 35S-labeled wt or msd PLP was translated in vitro for 15 min and incubated at 30°C for the time indicated. Then the samples were split in two, depleted of ATP and immunoprecipitated with anti-BiP (Figure 2C) or anti-calnexin. Co-immunoprecipitated PLP was quantified and expressed as a percentage of the total input. The graph shows the mean ± SEM of three independent experiments. (C) Stable cell lines were induced to express wt or msd PLPmh overnight in the presence or absence of 5 μg/ml BFA and then analysed as in (A). An eighth of the total lysate from induced wt and msd cells used for immunoprecipitation is shown in lanes 9 and 10. (D) Stable cell lines were induced to express msd PLPmh overnight (lanes 1–9) and HeLa cells transfected with opsin (lanes 10–15) were incubated with 10 μg/ml tunicamycin (Tun) or 0.5 mM castanospermine (Cas) for 6 h, and then analysed as in (A). An eighth of the total lysates used for immunoprecipitation are shown in lanes 7–9. (E) Stable cell lines were induced and immunoprecipitated as in (A), and then western blotted with the antibodies indicated. An eighth of the total lysates used for immunoprecipitation are shown in lanes 5 and 6. (F) Stable cell lines were left uninduced (−) or induced (+) and immunoprecipitated as in (A), and were then western blotted with anti-calreticulin (crt). An eighth of the total lysates used for immunoprecipitation are shown in lanes 5–8. Download figure Download PowerPoint Since calnexin is a relatively abundant integral membrane protein of the ER, we further investigated the specificity of its interaction with msd PLPmh by analyzing immuno-isolated PLPmh for other abundant ER membrane proteins (Figure 4E). We found no evidence for the association of wt or msd PLPmh with subunits of the Sec61 translocon or the SPC complex. Therefore we conclude that the association of msd PLPmh with calnexin reflects a specific interaction and is not due to the immunoprecipitation of protein aggregates, patches of ER membrane or similar phenomena (Cannon et al., 1996). We were unable to detect any association between msd PLPmh and calreticulin, the soluble equivalent of calnexin (Figure 4F). These two chaperones possess similar substrate specificities (Danilczyk et al., 2000) and have closely related structures apart from the TM domain which is present only in calnexin. This led us to suspect that calnexin may recognize and bind to the TM domains of misfolded PLP. Calnexin binds to the fourth transmembrane domain of PLP The msd mutation lies within TM4 of PLP and, although this mutation clearly affects the folding of the lumenal and cytosolic domains (as evidenced by the association of BiP and Hsc70 with msd PLP), we anticipated that the primary folding defect would be within TM4. Since calnexin, but not calreticulin, binds stably to msd PLPmh, we considered it likely that calnexin interacts with PLP via TM4. To test this hypothesis, we generated a truncated version of PLP encoding the fourth TM domain of PLP alone. To ensure that TM4 was properly inserted into the ER membrane, we fused the cleavable signal sequence from preprolactin to the N-terminus of TM4 and added a C-terminal ha tag to facilitate detection (Figure 5A). Figure 5.Calnexin binds the fourth TM domain of PLP. (A) Cartoon depicting the TM4ha construct. The preprolactin signal sequence is shown in gray, TM4ha is shown in black and the position of the msd mutation is indicated by an asterisk. (B) COS-7 cells were transiently transfected with wt (left) or msd (right) TM4ha, fixed with methanol and stained with anti-ha followed by a FITC-conjugated secondary antibody. Bar, 20 μm. (C) COS-7 cells were transiently transfected with msd TM4ha, fixed with 2% formaldehyde and 0.2% gluteraldehyde, permeabilized with 0.05% SDS (left column) or 40μg/ml digitonin (right column) and then stained with anti-ha (top row), anti-KDEL antibody 1D3 (center row) and DAPI (bottom row). Bar, 20 μm. (D) Lysates of COS-7 cells transiently expressing wt or msd PLPha and wt or msd TM4ha were western blotted with anti-ha. TM4 with (ssTM4ha) and without (TM4ha) the signal sequence present are indicated. (E) COS-7 cells transiently expressing wt or msd PLPha and wt or msd TM4ha were solubilized and immunoprecipitated with anti-ha or control (ctl) antibodies. Co-immunoprecipitated material was western blotted with anti-calnexin (top panel) or anti-BiP (bottom panel). (F) COS-7 cells transiently expressing msd TM4ha were solubilized and immunoprecipitated with anti-calnexin (cnx) or control (ctl) antibodies, and immunoprecipitated was material western blotted with anti-ha. The ratio of TM4ha to ssTM4ha in calnexin immunoprecipitates and total lysates was approximately 3.5:1 and 4.5:1, respectively. Download figure Download PowerPoint When expressed in COS-7 cells, the wt TM4ha and a version containing the msd mutation were localized to the ER (Figure 5B). In order to establish the transmembrane orientation of TM4ha, we examined the accessibility of the C-terminal ha epitope under different conditions. We found that TM4ha could be stained with anti-ha in digitonin permeabilized cells in which the ER membrane remains intact, as shown by the lack of staining with the lumenal marker 1D3 which was clearly visible if cells were permeabilized with SDS (Figure 5C). This confirmed that the ha epitope was on the cytoplasmic face of the ER membrane. Hence TM4ha was efficiently targeted to, and correctly oriented in, the ER membrane. Consistent with this, western blotting lysates from cells transiently transfected with TM4ha showed that the signal sequence was cleaved from the majority of the expressed protein (Figure 5D, TM4ha), although a small proportion still contained the signal sequence and ran with a slightly lower mobility (Figure 5D, ssTM4ha). We expressed msd TM4ha in vivo and found that this single transmembrane domain was able to co-immunoprecipitate calnexin with an efficiency comparable to that seen with the full-length msd PLPmh (Figure 5E, top panel). The lumenal chaperone BiP did not co-immunoprecipitate with wt or msd TM4ha (Figure 5E, bottom panel), confirming that TM4ha does not associate non-specifically with multiple ER chaperones. To confirm the interaction between TM4ha and calnexin, we performed the immunoprecipitation in reverse and showed that TM4ha was precipitated by anti-calnexin but not by a control antibody (Figure 5F). Importantly, calnexin co-immunoprecipitated the faster migrating TM4ha protein, showing that calnexin binds to the correctly oriented signal-sequence-cleaved TM4ha. The slower migrating ssTM4ha was also co-immunoprecipitated. It is likely that ssTM4 results from inefficient signal sequence cleavage, but it is formally possible that it has the opposite orientation and is also recognized by canexin as being 'misfolded'. These results provide the first direct evidence that calnexin can bind to isolated TM domains. Calnexin might also bind to TM1, TM2 and/or TM3 of PLP, but we have not tested this here. Interestingly, calnexin associated equally well with the 'wt' TM4ha and the version containing the msd mutation (Figure 5E, compare lanes 6 and 8), suggesting that calnexin recognizes an 'unassembled' TM domain of a polytopic protein as being misfolded. We speculate that in full-length PLP, the msd mutation may prevent the proper assembly of TM4 into the folde
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