A Membrane Protein Complex Mediates Retro-Translocation from the ER Lumen Into the Cytosol

articles A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol Yihong Ye1, Yoko Shibata1, Chi Yun2, David Ron2 & Tom A. Rapoport1 1Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA 2Skirball Institute, New York University School of Medicine, New York, New York 10016, USA ........................................................................................................................................................................................................................... Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor. Many proteins of eukaryotic cells undergo folding and modification the ER lumen and the function of p97 in the cytosol. Here we report in the lumen of the endoplasmic reticulum (ER). Properly folded the identification of a membrane protein complex that provides this polypeptides leave the ER along the secretory pathway, whereas link. misfolded proteins or unassembled protein complexes are retained1. These proteins are eventually degraded by the proteasome and must A membrane receptor for p97 therefore be transported back into the cytosol by a multi-step Previous experiments had shown membrane association of p97 and process called retro-translocation, dislocation, or ERAD (for ER- its cofactor Ufd1/Npl4 and suggested the existence of a membrane associated protein degradation) (for review, see ref. 2). Blocking receptor6. To identify the receptor, we first demonstrated that retro-translocation induces the unfolded protein response (UPR), a purified, recombinant p97 associates and co-sediments with iso- collection of signalling pathways that adapt cells to ER stress. The lated ER membranes from dog pancreas (Fig. 1a, lane 2). A p97 retro-translocation pathway has been co-opted by certain viruses to mutant lacking the amino-terminal domain (p97DN)6 interacted selectively destroy cellular proteins required for the immune defence only weakly (lane 4). The membrane interaction of p97 is not of the host. For example, the US11 protein of human cytomegalo- mediated by its cofactor Ufd1/Npl4. After washing the membranes virus (HCMV) targets newly synthesized major histocompatibility with high salt, Ufd1 was removed, while ,30% of the endogenous complex (MHC) class I heavy chains for retro-translocation3. These p97 remained membrane-bound (Fig. 1a, lane 10 versus 9). The observations, and the role played by ER stress in the pathophysiol- cofactor-depleted membranes still bound recombinant p97 (lane 6) ogy of important human diseases4, emphasize the need for an and again the p97DN had a much reduced affinity (lane 8). understanding of the mechanism of retro-translocation. Together, these results indicate that p97 binds directly to ER One of the best established components of the retro-translocation membranes, and that high affinity binding requires its N-domain. machinery is the cytosolic ATPase p97 (also called VCP or, in yeast, Protease pretreatment of the salt-washed membranes abolished the Cdc48), which interacts with a cofactor complex consisting of Ufd1 interaction (data not shown), suggesting that p97 binds to an and Npl4 (ref. 5). A retro-translocation substrate emerging on the integral membrane protein receptor. cytosolic side of the ER membrane is poly-ubiquitinated and We next labelled p97 with an amino-reactive biotin derivative, recognized by p97 (ref. 6). The ATPase probably then ‘pulls’ the containing a disulphide bridge between the reactive group and substrate out of the ER membrane by a mechanism similar to that biotin. The modified p97 protein was added to salt-washed ER proposed for AAA proteases in mitochondria and bacteria (for membranes, unbound material was removed by sedimentation, and review, see ref. 7). In yeast, mutations in constituents of the ATPase the membranes were solubilized in digitonin. The detergent extract complex block the degradation of all misfolded ER substrates was incubated with streptavidin beads and bound proteins were tested8–12. In mammals, the complex is required for the US11- eluted by reducing the disulphide linker. The Coomassie blue- mediated retro-translocation of MHC class I heavy chains6,9. stained gel showed two bands that were absent from the input Other steps in retro-translocation are less well understood. p97 preparation (Fig. 1b, lane 2 versus 3). Because staining intensity Substrate recognition must occur in the ER lumen. It appears that is approximately proportional to molecular mass, it seems that the US11 binds specifically to MHC class I heavy chains13 and that two proteins are present in near-stoichiometric amounts compared certain ER chaperones recognize misfolded proteins12,14–18, but how with membrane-bound p97. These proteins were not seen when these substrates are subsequently targeted to the retro-translocation p97DN was used, even though a small amount of p97DN bound to machinery and cross the ER membrane is unclear. Some evidence the membrane (lane 1). Sequencing by mass spectrometry gave suggests that they may be transported through the Sec61 channel, several peptides that allowed unambiguous identification of the which is responsible for transporting proteins from the cytosol into proteins (Supplementary Table 1). the ER, but the data are inconclusive (for discussion, see ref. 2). The upper band corresponds to a homologue of S. cerevisiae Der1 Importantly, there is as yet no link between substrate recognition in (for ‘degradation in the ER’), a protein identified in a genetic screen 841 NATURE | VOL 429 | 24 JUNE 2004 | www.nature.com/nature © 2004 Nature Publishing Group articles for components required for the degradation of a misfolded luminal were co-precipitated (Fig. 1d, lanes 2, 3). No precipitation was ER protein19. Essentially nothing is known about Der1’s function, observed with a pre-immune serum (lane 1) or with an antiserum except that it is required for the degradation of other substrates20, depleted of VIMP antibodies (lane 4). When Myc-tagged VIMP and and that its expression is upregulated under ER stress21. We call the p97 were co-expressed in HEK293 cells, the two proteins could be Der1 homologue Derlin-1 to indicate that it is Der1-like. Homo- co-immunoprecipitated with Myc antibodies (Supplementary logues of Derlin-1 are found in every eukaryotic organism (Fig. 1c; Fig. S4). A similar result was obtained with p97AA, a mutant 6 Supplementary Fig. S1). All species contain at least one other related defective in substrate binding , indicating that VIMP does not protein, called Derlin-2, that belongs to a distinct group (Fig. S1). It serve as substrate for p97 (Supplementary Fig. S4). p97DN did is unclear which of the two classes of Der1 homologues is more not co-precipitate with Myc-VIMP.Together, these data suggest that closely related to yeast Der1 (E. Hartmann, personal communi- Derlin-1 and VIMP form a membrane protein complex that serves cation). Mammalian Derlin-1 is predicted to have four trans- as a receptor for p97. membrane segments with both the amino and carboxy termini in the cytosol (Fig. 1c), consistent with experimental results on yeast VIMP links Derlin-1 with the p97 ATPase complex Der1 (ref. 22). Given that VIMP has a sizeable cytosolic domain, we suspected that The smaller p97-interacting protein does not have identifiable it may be responsible for the interaction with p97. Indeed, a purified homologues outside vertebrates. It had previously turned up in a 23 fusion protein containing glutathione S-transferase (GST) and the screen for selenocysteine-containing proteins , and is predicted to cytosolic domain of VIMP (GST–VIMPc) bound recombinant p97 span the membrane once, with a short luminal segment and a longer (data not shown). Interestingly, when GST-VIMPc was added to a cytosolic domain of ,132 amino acids (Supplementary Fig. S2). On solution of p97, a complex containing both proteins precipitated the basis of additional data (see below), we have named this protein (Fig. 2a, lanes 7–12). His-tagged VIMP also precipitated p97 (data VIMP: for VCP (another name for p97)-interacting membrane not shown). Because both p97 and VIMP are able to form oligomers protein (Gene bank ID: AY618665). (ref. 24, and Supplementary Fig. S5), precipitation is probably To confirm the interactions between Derlin-1, VIMP,and p97, we caused by extensive multivalent interactions, analogous to those used peptide-specific antibodies to human Derlin-1 (hDerlin-1) between a polyclonal antibody and its antigen. and VIMP (see Supplementary Fig. S3). A detergent extract of HeLa To test whether VIMP can bind p97 together with its cofactor

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