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The Role of a Novel p97/Valosin-containing Protein-interacting Motif of gp78 in Endoplasmic Reticulum-associated Degradation*

  • Petek Ballar
    Footnotes
    Affiliations
    Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201

    Program in Molecular Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Yuxian Shen
    Affiliations
    The Institute of Clinical Pharmacology, Anhui Medical University, Hefei, P. R. China
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  • Hui Yang
    Affiliations
    Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201
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  • Shengyun Fang
    Correspondence
    To whom correspondence should be addressed: Medical Biotechnology Center, University of Maryland Biotechnology Institute, UMBI Bldg., N359, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-2220; Fax: 410-706-8184;
    Affiliations
    Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201

    Program in Molecular Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant R01 GM69967 (to S. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
    1 Supported in part by a Fellowship from the Council of Higher Education of Republic of Turkey to the University of Ege.
Open AccessPublished:September 20, 2006DOI:https://doi.org/10.1074/jbc.M603355200
      Improperly folded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation, a process that dislocates misfolded proteins from the ER membrane into the cytosol, where they undergo proteasomal degradation. Dislocation requires a subclass of ubiquitin ligases that includes gp78 in addition to the AAA ATPase p97/VCP and its cofactor, the Ufd1-Npl4 dimer. We have previously reported that gp78 interacts directly with p97/VCP. Here, we identify a novel p97/VCP-interacting motif (VIM) within gp78 that mediates this interaction. We demonstrate that the VIM of gp78 recruits p97/VCP to the ER, but has no effect on Ufd1 localization. We also show that gp78 VIM interacts with the ND1 domain of p97/VCP that was shown previously to be the binding site for Ufd1. To evaluate the role of Ufd1 in gp78-p97/VCP-mediated degradation of CD3δ, a known substrate of gp78, RNA interference was used to silence the expression of Ufd1 and p97/VCP. Inhibition of p97/VCP, but not Ufd1, stabilized CD3δ in cells that overexpress gp78. However, both p97/VCP and Ufd1 appear to be required for CD3δ degradation in cells expressing physiological levels of gp78. These results raise the possibility that Ufd1 and gp78 may bind p97/VCP in a mutually exclusive manner and suggest that gp78 might act in a Ufd1-independent degradation pathway for misfolded ER proteins, which operates in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.
      The endoplasmic reticulum (ER)
      The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; E3, ubiquitin-protein isopeptide ligase; VIM, VCP-interacting motif; VIMP, VCP-interacting membrane protein; SVIP, small p97/VCP-interacting protein; CMV, cytomegalovirus; aa, amino acid(s); GST, glutathione S-transferase; HA, hemagglutinin; CHX, cycloheximide; siRNA, small interference RNA; wt, wild type; RNAi, RNA interference.
      3The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; E3, ubiquitin-protein isopeptide ligase; VIM, VCP-interacting motif; VIMP, VCP-interacting membrane protein; SVIP, small p97/VCP-interacting protein; CMV, cytomegalovirus; aa, amino acid(s); GST, glutathione S-transferase; HA, hemagglutinin; CHX, cycloheximide; siRNA, small interference RNA; wt, wild type; RNAi, RNA interference.
      is the first compartment in the secretory pathway (
      • Federovitch C.M.
      • Ron D.
      • Hampton R.Y.
      ,
      • Levine T.
      • Rabouille C.
      ). Proteins destined for the secretory pathway are translocated into the lumen or inserted into the membrane of the ER where they are properly folded and modified before being delivered to their functional destinations (
      • Federovitch C.M.
      • Ron D.
      • Hampton R.Y.
      ,
      • Levine T.
      • Rabouille C.
      ). However, protein folding in the secretory pathway is an imperfect process that generates misfolded products (
      • Hampton R.Y.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ). These misfolded proteins are retained in the ER by a qualitycontrol mechanism and eliminated through a process known as ER-associated degradation (ERAD) (
      • Hampton R.Y.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ,
      • Ahner A.
      • Brodsky J.L.
      ,
      • Romisch K.
      ). ERAD requires retrotranslocation, in which misfolded proteins are dislocated from the ER membrane into the cytosol where they are degraded by the proteasomes (
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ). Genetic evidence from yeast indicates that the ATPase CDC48 in combination with its cofactors, the Ufd1-Npl4 dimer, are essential for retrotranslocation (
      • Bays N.W.
      • Hampton R.Y.
      ). The mammalian homolog of CDC48, p97/VCP, in concert with Ufd1-Npl4 dimer, appears to play a similar role in mammalian cells (
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ,
      • Alzayady K.J.
      • Panning M.M.
      • Kelley G.G.
      • Wojcikiewicz R.J.
      ).
      p97/VCP, Ufd1, and Npl4 are cytosolic proteins. They must be recruited to the cytosolic surface of the ER to carry out the retrotranslocation process. In yeast, an ER membrane-anchored protein, Ubx2, plays a central role in coupling ubiquitination with retrotranslocation. Ubx2 recruits CDC48 along with the Ufd1-Npl4 dimer using its Ubx domain (
      • Neuber O.
      • Jarosch E.
      • Volkwein C.
      • Walter J.
      • Sommer T.
      ,
      • Schuberth C.
      • Buchberger A.
      ), thereby linking the CDC48-Ufd1-Npl4 complex to ER-anchored ERAD E3s, that include Hrd1 and Doa10, as well as ERAD substrates (see a diagrammatic illustration of this complex in Fig. 1a) (
      • Neuber O.
      • Jarosch E.
      • Volkwein C.
      • Walter J.
      • Sommer T.
      ,
      • Schuberth C.
      • Buchberger A.
      ). Recent studies suggest that more complicated mechanisms exist for coupling ubiquitination with retrotranslocation in higher eukaryotes (Fig. 1b) (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Schulze A.
      • Standera S.
      • Buerger E.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Koning F.
      • Kloetzel P.M.
      • Seeger M.
      ,
      • Oda Y.
      • Okada T.
      • Yoshida H.
      • Kaufman R.J.
      • Nagata K.
      • Mori K.
      ). In mammalian systems, p97/VCP interacts with a protein complex that contains at least four ER membrane-anchored proteins, the p97/VCP-interacting membrane protein (VIMP), Derlin-1, Derlin-2, and ERAD E3 Hrd1. The Ufd1-Npl4 dimer is also recruited along with p97/VCP by the four-protein complex (Fig. 1b) (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). This is consistent with previous reports that the p97/VCP-Ufd1-Npl4 complex is functionally required for ERAD in mammalian cells (
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ,
      • Alzayady K.J.
      • Panning M.M.
      • Kelley G.G.
      • Wojcikiewicz R.J.
      ). gp78, a multimembrane-spanning ERAD E3, also interacts with p97/VCP (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ,
      • Song B.L.
      • Sever N.
      • DeBose-Boyd R.A.
      ). Functionally, the gp78-p97/VCP interaction enhances the binding of p97/VCP to polyubiquitinated proteins, as well as degradation of ERAD substrates, including CD3δ, apolipoprotein B100, and 3-hydroxy-3-methylglutaryl-CoA reductase (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ,
      • Song B.L.
      • Sever N.
      • DeBose-Boyd R.A.
      ,
      • Liang J.S.
      • Kim T.
      • Fang S.
      • Yamaguchi J.
      • Weissman A.M.
      • Fisher E.A.
      • Ginsberg H.N.
      ). However, the role of p97/VCP cofactors, Ufd1 and Npl4, in gp78-mediated ERAD is yet to be determined.
      Figure thumbnail gr1
      FIGURE 1Current models of protein complexes that links ubiquitination to retrotranslocation during ERAD. a, in yeast the ER-anchored Ubx2 (labeled with x2) recruits a CDC48-Ufd1-Npl4 complex from the cytosol to the ER, thereby linking the CDC48 complex to E3 (Hrd1 and Doa10), ubiquitinated ERAD substrate (black rectangle), and Der1. This Ubx2-assembled complex couples ubiquitination with retrotranslocation and leads to degradation of ERAD substrates. U represents ubiquitin. b, in mammals, four ER-anchored proteins, including the E3 Hrd1, Derlin-1 and -2 (behind major histocompatibility complex class I heavy chain: HC in the black rectangle), and VIMP (cylinder behind E3), all interact with p97/VCP. The interaction leads to recruitment of the p97/VCP-Ufd1-Npl4 complex from the cytosol to the ER, thereby linking p97/VCP complex to Hrd1, substrate (HC), Derlin-1 and -2, and VIMP to couple ubiquitination with retrotranslocation.
      In this study, we have identified a novel p97/VCP-interacting motif (VIM) in the cytosolic tail of gp78 and in the N-terminal region of the small p97/VCP-interacting protein (SVIP) (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). The VIM of gp78 interacts with the ND1 domain of p97/VCP, the reported binding site for SVIP, Ufd1, and p47, another p97/VCP cofactor required for Golgi and ER membrane fusion (
      • Uchiyama K.
      • Jokitalo E.
      • Kano F.
      • Murata M.
      • Zhang X.
      • Canas B.
      • Newman R.
      • Rabouille C.
      • Pappin D.
      • Freemont P.
      • Kondo H.
      ,
      • Wang Y.
      • Satoh A.
      • Warren G.
      • Meyer H.H.
      ). SVIP, Ufd1, and p47 are known to bind p97/VCP in a mutually exclusive manner. In the current study, our data suggest that gp78 and Ufd1 also bind p97/VCP in a mutually exclusive fashion. Functionally, gp78-mediated ERAD apparently requires p97/VCP but not Ufd1. We have also confirmed the presence of an Ufd1-dependent ERAD pathway. Collectively, these data suggest that gp78 may mediate an Ufd1-independent degradation pathway for misfolded ER proteins. This pathway appears to operate in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.

      EXPERIMENTAL PROCEDURES

      Plasmids and Antibodies—Mouse Ufd1 with an N-terminal FLAG tag was cloned into the pcDNA3.1 vector to generate pcDNA3-FLAG-Ufd1. To construct pFLAG-His-Npl4, His-Npl4 cDNA was excised from pQE9-Npl4 (generously provided by Dr. Chou-chi Li, NCI-Frederick) and then cloned into the pFLAG-CMV vector. The pFLAG-CMV-N (aa 1-198), ND1 (aa 1-470), D1 (aa 199-470), and D2 (aa 471-806) domains of mouse p97/VCP were generated based on a recent structural study (
      • DeLaBarre B.
      • Brunger A.T.
      ). pcDNA3-gp78 (mouse) was a gift from Dr. Jun Yotoka (National Cancer Center Research Institute, Japan). pcDNA3-gp78ΔVIM was created by site-directed mutagenesis to introduce a stop codon that truncates the C-terminal 30 amino acids (VIM). To make recombinant GST-VIM, cDNA encoding the VIM of human gp78 was cloned into the pGEX4T3 vector (pGEX4T3-VIM). pCIneo-Hrd1-FLAG has been previously reported (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). A pCIneo-Hrd1-VIM-FLAG chimera was generated by inserting cDNA encoding the VIM of mouse gp78 through a SacII site between the C terminus of Hrd1 and the FLAG tag of pCIneo-Hrd1-FLAG (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). pCI-HA-CD3δ has been previously described (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). Monoclonal anti-Ufd1 antibody was generously provided by Dr. Hemmo Meyer (Swiss Federal School of Technology, Switzerland) and purchased from BD Biosciences. Monoclonal anti-gp78 antibody (clone 2G5) was generated using recombinant GST-gp78C as antigen. 2G5 recognizes the epitope located between amino acids 497-578 of gp78.
      Fractionation, Immunoprecipitation, and Immunoblotting—For subcellular fractionation, 293 cells were transfected as indicated in the figure legends. Cell fractionation was carried out by homogenization in fractionation buffer containing 50 mm Tris-HCl (pH 8), 1 mm 2-mercaptoethanol, 1 mm EDTA, 10 mm triethanolamine, and 0.32 m sucrose via passage through a 27-gauge syringe 20 times. Homogenates were centrifuged at 1000 × g to sediment unbroken cells, cell debris, and nuclei (pellet fraction (p)). The remaining supernatant was separated by centrifugation at 105,000 × g for 60 min. The resulting pellet contained the microsomal fraction (m) and the supernatant contained the cytosolic fraction (c). All three fractions (m, c, and p) were further processed for immunoblotting. Immunoprecipitation and immunoblotting were carried out following previously published protocols (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ).
      Interaction between GST-VIM and Purified p97/VCP—His-tagged p97/VCP, Ufd1, and Npl4 were expressed in E. coli after induction with isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested and lysed by sonication in buffer containing 500 mm KCl, 100 mm Tris/HCl (pH 7.4). 5 mm MgCl2, 1 mm ATP, 4 mm dithiothreitol, 20 mm imidazole. His-tagged p97/VCP, Ufd1, and Npl4 in lysates were affinity-purified using nickel-nitrilotriacetic acid-agarose beads (Qiagen) and eluted with lysis buffer containing 300 mm imidazole. Imidazole was removed by dialysis against 1× phosphate-buffered saline containing 1 mm dithiothreitol. The in vitro binding assay was carried out essentially as previously reported (
      • Meyer H.H.
      • Shorter J.G.
      • Seemann J.
      • Pappin D.
      • Warren G.
      ). Briefly, 3 μg of GST-VIM immobilized and purified on glutathione agarose beads was incubated with His-tagged p97/VCP, Ufd1, and Npl4 proteins (2 μg each) for 1 h at 4°C in binding buffer containing 25 mm Tris/HCl (pH 8.0), 200 mm KCl, 2 mm MgCl2, 1 mm ATP, 1mm dithiothreitol, 5% glycerol, and 1% Triton X-100. After washing, GST-VIM-bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining.
      Fluorescence Microscopy—U2OS cells were cultured on coverslips in 6-well dishes and transfected with the indicated plasmids using Lipofectamine 2000. For double immunofluorescent staining, gp78 and endogenous p97/VCP were labeled using rabbit anti-gp78 and mouse anti-p97/VCP antibodies in conjunction with Alexa Fluor-594 and Alexa Fluor-488. A similar procedure was used to label gp78 and Ufd1, as well as Hrd1/Hrd1 plus VIM and p97/VCP. Staining was performed as described previously (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). Fluorescent microscopy was performed using a Zeiss Axiovert 200M fluorescent microscope.
      Establishment of 293 Cell Lines That Stably Express HA-CD3δ or HA-CD3δ and gp78—293 cells were transfected with plasmids encoding HA-CD3δ or HA-CD3δ and gp78. pBABE vector conferring puromycin-resistance was co-transfected for stable clone selection. Twenty-four hours after transfection 2.5 μg/ml puromycin was added to eliminate nontransfected cells. Culture medium was refreshed every 3 days until cell colonies became evident. Positive clones expressing HA-CD3δ (CD3δ clones) or HA-CD3δ plus gp78 (CD3δ/gp78 clones) were characterized by immunoblotting and cycloheximide (CHX) chase analysis. CHX chase was performed as previously described (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ).
      RNA Interference—pSUPER-gp78 siRNA1 was used to knockdown endogenous gp78 expression as previously reported (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). Silencer ® pre-designed siRNAs target p97/VCP and Ufd1, as well as serves as a negative control, were purchased from Ambion (Austin, TX). CD3δ clone-5 and CD3δ/gp78 clone-20 cells were transfected with appropriate siRNA(s) using Lipofectamine 2000. 72 h after transfection cells were harvested and processed for further experiments.
      Pulse-chase Analysis—Pulse-chase analysis was performed essentially as we have previously described (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). CD3δ clone-5 and CD3δ/gp78 clone-20 cells with a knockdown of either p97/VCP or Ufd1 were pulse-labeled with a mixture of l-[35S]methionine and l-[35S]cysteine (Redivue™ Pro-mix l-[35S] in vitro cell labeling mix, GE HealthCare) at a concentration of 200 μCi/ml for 30 min. After labeling, unincorporated l-[35S]methionine and l-[35S]cysteine were removed and chased for 0, 2, 4, and 6 h. Labeled cells were processed for immunoprecipitation of HA-tagged CD3δ followed by SDS-PAGE. Radioactive CD3δ was detected by a Typhoon scanner (Amersham Biosciences) and quantified by Image-QuaNT (Molecular Dynamics).

      RESULTS

      Identification of a Novel p97/VIM in gp78 and SVIP—Many p97/VCP-interacting proteins have been reported (
      • Dreveny I.
      • Pye V.E.
      • Beuron F.
      • Briggs L.C.
      • Isaacson R.L.
      • Matthews S.J.
      • McKeown C.
      • Yuan X.
      • Zhang X.
      • Freemont P.S.
      ,
      • Wang Q.
      • Song C.
      • Li C.C.
      ,
      • Elsasser S.
      • Finley D.
      ). In search of a common p97/VCP-interacting domain that might be present in different p97/VCP-interacting proteins, we identified a conserved amino acid sequence within the C-terminal portion of gp78 and the N-terminal region of SVIP, both of which have previously been found to bind p97/VCP (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ,
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). We named this sequence the p97/VCP-interacting motif (VIM). VIM comprises the C-terminal 30 amino acids of gp78 and is well conserved in vertebrates (examples are given in Fig. 2a). However, it is not present in the yeast Cue1p protein or Caenorhabditis elegans gp78. Consistently, Cue1p and C. elegans gp78 do not interact with p97/VCP (supplemental Fig. S1). Bioinformatic analysis predicts a helix formed by 17 amino acids (aa 625-641 in gp78 and aa 20-35 in SVIP, underscored in Fig. 2a) within the VIM of human gp78 and SVIP (
      • Rost B.
      • Yachdav G.
      • Liu J.
      ). To ascertain whether the VIM of gp78 is sufficient to bind p97/VCP, we assessed its ability to recruit p97/VCP to microsomal membranes. 293 cells were transfected with wt gp78 or its VIM deletion mutant (gp78ΔVIM) (Fig. 2b). Transfected cells were fractionated into cytosol (c) and ER-containing microsomal membrane (m) fraction where gp78 is generally localized, which we have previously reported (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). Wild-type (wt) gp78 efficiently moves p97/VCP from the cytosol to the microsomes as determined by immunoblotting (Fig. 2, b and c, lanes 4 and 5 versus lanes 1 and 2). Deletion of VIM from gp78 (gp78ΔVIM) largely diminishes this effect (Fig. 2b, lanes 10 and 11 versus lanes 4 and 5). To investigate the role of the E3 activity of gp78 in relocalization of p97/VCP, we examined the effects of gp78R2m on p97/VCP localization in cytosol and microsomal fractions. gp78R2m is an E3-inactive mutant form of gp78 with two defective zinc ligand residues in the RING finger (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). The results showed that gp78R2m recruits p97/VCP from the cytosol to the microsomes (Fig. 2b, lanes 7 and 8 versus lanes 1 and 2) as efficiently as wt gp78 (Fig. 2b, lanes 4 and 5 versus lanes 7 and 8). These results strongly suggest that the VIM of gp78 interacts with and recruits p97/VCP from the cytosol to the ER and gp78 E3 activity is not required for p97/VCP relocalization.
      Figure thumbnail gr2
      FIGURE 2Identification of a p97/VCP-interacting motif (VIM) in gp78 and SVIP. a, alignments of the VIM of gp78 and SVIP. Hs, human; Rn, rat; Mm, mouse; aa, amino acid. Underscored is the predicted sequence that forms a helix. b, 293 cells were transfected with plasmids encoding wt gp78, or gp78ΔVIM, or gp78R2m. Empty vector was transfected as a negative control. Eighteen hours after transfection, cells were fractionated into membrane (m) and cytosol (c). The initial pellet (p) obtained from clearing the homogenates was retained to monitor total levels of proteins. Endogenous p97/VCP was revealed by immunoblotting using monoclonal antibody. p97/VCP in the microsome (m) and cytosolic (c) fractions are framed for easy comparison. Transfected gp78 was detected by immunoblotting using polyclonal anti-gp78 antibodies that do not recognize endogenous gp78 as previously reported (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). gp78 tends to form high molecular weight smears (arrow) due to oligomerization and autoubiquitination (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). *, residual signal of p97/VCP from the first blotting for p97/VCP. Calnexin was blotted as an ER marker. c, 293 cells were transfected with plasmids encoding wt gp78 or empty vector as control. Transfected cells were analyzed as in b. Endogenous p97/VCP and Ufd1 were blotted using monoclonal antibodies.
      A previous study has shown that VIMP, another ER-anchored p97/VCP-interacting membrane protein, co-localizes with p97/VCP in the ER (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). Importantly, VIMP interacts with p97/VCP along with its cofactor, the Ufd1-Npl4 dimer (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). To determine whether gp78 could recruit p97/VCP along with Ufd1, a similar fractionation experiment to that shown in Fig. 2b was performed and the effects of gp78 on localization of endogenous Ufd1 were examined by immunoblotting. According to this data, gp78 has no apparent effect on Ufd1 localization despite the fact that p97/VCP is relocalized to the microsomes (Fig. 2c, lanes 1 and 2 versus lanes 3 and 4). These results suggest that gp78 interacts only with p97/VCP and that Ufd1 is not a part of the gp78-p97/VCP complex (Fig. 2c, lanes 1 and 2 versus lanes 4 and 5).
      Based on previous studies showing that SVIP, Ufd1, and p47 bind to the ND1 domain of p97/VCP in a mutually exclusive manner (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ), we reasoned that gp78 might also bind to the p97/VCP ND1 domain. A GST pull-down assay with GST fusion of the VIM of gp78 (GST-VIM) was designed to test this hypothesis. Various truncation mutants of p97/VCP (N (aa 1-198), ND1 (aa 1-470), D1 (aa 199-470), and D2 (aa 471-806) domain) tagged with FLAG were generated based on structural studies that pinpointed key structural domains of p97/VCP (Fig. 3a) (
      • DeLaBarre B.
      • Brunger A.T.
      ). In pull-down assays with 293 cell lysates, the VIM of gp78 interacted only with proteins containing the ND1 domain of p97/VCP (Fig. 3b, lanes 1 and 2). Therefore, like Ufd1, p47, and SVIP, gp78 also binds to the ND1 domain of p97/VCP.
      Figure thumbnail gr3
      FIGURE 3The VIM of gp78 interacts with the ND1 domain of p97/VCP. a, diagrammatic representation of p97/VCP and its mutants with N-terminal FLAG (FL) tag. b, GST-VIM interacts with the ND1 domain of p97/VCP. Lysates from 293 cells transfected with FLAG-tagged p97/VCP or FLAG-tagged N, ND1, D1, or D2 domain of p97/VCP were used for pull-down assays with GST-VIM from gp78. Only the full-length p97/VCP and its ND1 domain were pulled down by GST-VIM (lanes 1 and 2). Whole cell lysates (WCL) used for pull-down are shown in lanes 6-10. The lower panel shows Coomassie Blue staining of GST-VIM purified on glutathione agarose-beads used in the pull-down assay.
      GST-VIM Interacts with p97/VCP but Not the p97/VCP-Ufd-Npl4 Complex—The interaction of gp78 with the ND1 domain of p97/VCP suggests that gp78 and Ufd1 might form mutually exclusive complexes with p97/VCP. To test this possibility, GST-VIM pull-down assays of lysates from 293 cells transfected with FLAG-tagged Ufd1 and FLAG-His-tagged Npl4 were conducted. In these experiments, p97/VCP alone was associated with GST-VIM but not Ufd1-Npl4 or complexes between p97/VCP and Ufd1-Npl4 (Fig. 4a, lanes 4 and 12). Overexposing the blot did not reveal any additional bands (data not shown). These results further support the notion that the VIM of gp78 mediates the formation of the gp78 and p97/VCP complex from which Ufd1 is excluded. However, a second possibility also exists: p97/VCP might not be able to form a complex with Ufd1-Npl4 dimer under the conditions we used for the pull-down assay shown in Fig. 4a. To distinguish between the two possibilities, we carried out a pull-down of Npl4 with nickel-nitrilotriacetic acid-agarose beads. 293 cells were transfected with plasmids encoding FLAG-Ufd1, FLAG-His-Npl4, or both (Fig. 4b, lanes 1-4). Lysates prepared from the transfected cells were used for a pull-down assay with nickel-nitrilotriacetic acid-agarose beads. Analysis of the precipitates by immunoblotting indicates that, indeed, p97/VCP, Ufd1, and Npl4, appear to be capable of forming a complex (Fig. 4b, lane 8) under conditions used for the GST-pull-down assay (Fig. 4a). Therefore, it is plausible that GST-VIM forms a complex with p97/VCP and excludes Ufd1-Npl4 from the complex.
      Figure thumbnail gr4
      FIGURE 4GST-VIM interacts with p97/VCP, but not the p97/VCP-Ufd1-Npl4 complex. a, GST-VIM pulls down p97/VCP, but not p97/VCP-Ufd1-Npl4 complex. Lysates prepared from 293 cells transfected with FLAG-Ufd1 and FLAG-6His-Npl4 were used for GST-VIM pull-down. p97/VCP, but not Ufd1 and Npl4, were pulled down by GST-VIM (lanes 9-12). GST alone pulls down neither p97/VCP nor Ufd1 or Npl4 (lanes 5-8). Whole cell lysates (WCL) used for the pull-down assay are shown in lanes 1-4. b, p97/VCP, FLAG-Ufd1, and FLAG-6His-Npl4 form complex in cells. 293 cells transfected as in a were lysed and used for nickel-nitrilotriacetic acid-agarosebead pull-down of FLAG-6His-Npl4. p97/VCP and FLAG-Ufd1 are associated with FLAG-6His-Npl4 (lane 8). WCL used for pull-down is shown in lanes 1-4. c, GST-VIM of gp78 interacts with purified recombinant p97/VCP. Two micrograms each of purified His-tagged p97/VCP, Ufd1, and Npl4 were incubated with 3 μg of GST-VIM or GST immobilized on beads. After washing, GST-VIM-bound proteins were detected by Coomassie Blue staining. Lanes 1, molecular weight marker; 2, 6His-p97/VCP; 3, 6His-Npl4; 4, 6His-Ufd1; 5, GST-VIM alone; 6, GST-VIM plus 6His-p97/VCP plus 6His-Ufd1 plus 6His-Npl4; 7, GST plus 6His-p97/VCP plus 6His-Ufd1 plus 6His-Npl4. d, schematic illustration of results from a to c. p97/VCP forms a mutually exclusive complex with GST-VIM and Ufd1-Npl4 dimer.
      Next, we sought to determine whether GST-VIM could directly interact with p97/VCP. Purified recombinant p97/VCP, Ufd1, and Npl4 from bacteria were co-incubated with GST-VIM immobilized glutathione-agarose beads. The results show that GST-VIM selectively binds p97/VCP despite the presence of Ufd1 and Npl4 (Fig. 4c, lane 6). Importantly, the purified p97/VCP, Ufd1, and Npl4 appear to be able to form a complex under the same conditions in the absence of gp78 (data not shown). Together, these data suggest that gp78 and Ufd1-Npl4 dimer form mutually exclusive complexes with p97/VCP in cells (Fig. 4d).
      gp78 Recruits p97/VCP but Not Ufd1 Complex to the ER through Its VIM—We have previously reported that gp78 is largely localized in the ER based on the fact that it co-localizes with the ER-anchored ERAD E2 MmUbc6 (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Tiwari S.
      • Weissman A.M.
      ). In this report, we have demonstrated that gp78 can recruit p97/VCP from the cytosol to microsomes (Fig. 2, b and c). Here, we asked whether gp78 could recruit p97/VCP to the ER in intact cells. Using double immunofluorescent staining, we examined the effects of gp78 and gp78ΔVIM on the localization of endogenous p97/VCP. U2OS cells were chosen for these experiments because their flattened morphology allows excellent imaging of the ER network (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). A plasmid construct encoding wt gp78 or gp78ΔVIM was transfected into U2OS cells and cells were double-stained with a rabbit polyclonal anti-gp78 (recognizing both gp78 and gp78ΔVIM) and mouse monoclonal anti-p97/VCP. Cells transfected with empty vector exhibited diffuse distribution of endogenous p97/VCP in the cytosol and nuclei (Fig. 5a). Wt gp78 expression relocalizes p97/VCP from the cytosol to the ER where gp78 resides (Fig. 5b). Deletion of VIM from gp78 (gp78ΔVIM) markedly reduces this effect (Fig. 5c), suggesting that gp78 recruits p97/VCP to the ER via its VIM.
      Figure thumbnail gr5
      FIGURE 5The VIM of gp78 mediates relocalization of p97/VCP from the cytosol to the ER in cells. a, localization of endogenous p97/VCP in control cells. p97/VCP was detected by indirect immunofluorescent staining with monoclonal anti-p97/VCP. b and c, plasmid encoding wt gp78 or gp78ΔVIM was transfected into U2OS cells. Endogenous p97/VCP and transfected wt gp78 and gp78ΔVIM were stained by indirect double immunofluorescent labeling using mouse anti-p97/VCP (left panels) and rabbit anti-gp78 (middle panels) antibodies. Merged images are shown in the right panels. d and e, plasmid encoding wt Hrd1-FLAG or Hrd1 plus VIM-FLAG was transfected into U2OS cells. Transfected cells were stained with monoclonal anti-p97/VCP (left panels) and polyclonal anti-FLAG (middle panels). Merged images are shown in the right panels. f, wt gp78 was co-transfected with FLAG-Ufd1 into U2OS cells. Double immunofluorescent staining was performed for FLAG (label FLAG-Ufd1, left panel) and gp78 (middle panel). The right panel shows the merged image.
      To test this possibility further, we generated a construct that expresses a fusion protein of VIM to the C terminus of Hrd1, another ER-anchored E3. Double immunofluorescent staining revealed that Hrd1 alone has minimal effects on the localization of p97/VCP (Fig. 5d). However, fusion of VIM to Hrd1 (Hrd1 plus VIM) dramatically increases co-localization of p97/VCP with Hrd1 plus VIM (Fig. 5e). Interestingly, the co-localized p97/VCP and Hrd1 plus VIM exhibit a punctate pattern in the cytoplasm of the cells. Because Hrd1 is an ER protein, the punctate structures are likely to be derived from the ER. These results further indicate that the VIM of gp78 mediates the recruitment of p97/VCP from the cytosol to the ER.
      gp78 recruits p97/VCP but does not appear to recruit Ufd1 to the microsomes by fractionation assay (Fig. 2c). To confirm this unexpected result, immunofluorescent staining was utilized to further test the effect of gp78 on Ufd1 localization in cells. We co-transfected U2OS cells with plasmids that encoded FLAG-tagged Ufd1 and wt gp78. Transfected cells were doubly labeled with monoclonal anti-FLAG antibody for Ufd1 and polyclonal anti-gp78 antibodies. Consistent with the fractionation results (Fig. 2c), we found that gp78 does not affect Ufd1 localization in cells (Fig. 5f). Together, these results suggest that gp78 recruits p97/VCP but not the p97/VCP-Ufd1-Npl4 complex to the ER.
      Ufd1 Is Dispensable in gp78-mediated Degradation of CD3δ—To analyze the involvement of p97/VCP and its cofactor Ufd1-Npl4 dimer in the gp78-mediated ERAD of CD3δ, we established 293 cell lines that stably express CD3δ and gp78 (generating CD3δ/gp78 clones, Fig. 6, lanes 7-12) or CD3δ alone (our control: CD3δ clones, Fig. 6, lanes 1-6). In both cell lines, CD3δ was rapidly degraded as revealed by CHX chase analysis, a technique widely used in determining degradation of ERAD substrates in both yeast and mammalian cells (
      • Gardner R.G.
      • Shearer A.G.
      • Hampton R.Y.
      ,
      • Medicherla B.
      • Kostova Z.
      • Schaefer A.
      • Wolf D.H.
      ,
      • Ando H.
      • Watabe H.
      • Valencia J.C.
      • Yasumoto K.
      • Furumura M.
      • Funasaka Y.
      • Oka M.
      • Ichihashi M.
      • Hearing V.J.
      ,
      • Sharma M.
      • Pampinella F.
      • Nemes C.
      • Benharouga M.
      • So J.
      • Du K.
      • Bache K.G.
      • Papsin B.
      • Zerangue N.
      • Stenmark H.
      • Lukacs G.L.
      ,
      • Chen B.
      • Mariano J.
      • Tsai Y.C.
      • Chan A.H.
      • Cohen M.
      • Weissman A.M.
      ). Steady-state levels of CD3δ were lower in CD3δ/gp78 clones (Fig. 6, lanes 1-6 versus 7-12), which may be due to enhanced degradation of CD3δ associated with gp78 overexpression. The level of gp78 is approximately four times higher in CD3δ/gp78 clones than that in CD3δ clones (Fig. 6, lanes 7-12 versus 1-6). gp78 levels also decrease during a CHX chase (Fig. 6). This is consistent with our previous report that gp78 is an unstable protein degraded through ERAD (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ).
      Figure thumbnail gr6
      FIGURE 6Establishment of 293 cell lines that stably express CD3δ or CD3δ and gp78. 293 cells were transfected with plasmids encoding HA-CD3δ or HA-CD3δ and gp78. pBABE vector encoding puromycin-resistant gene was co-transfected for stable clone selection. Examples of positive clones expressing HA-CD3δ (CD3δ clones) or HA-CD3δ plus gp78 (CD3δ/gp78 clones) were characterized by immunoblotting and CHX chase analysis. The arrow denotes full-length gp78. The bands and smear above the full-length gp78 (arrow) represent its oligomerized and autoubiquitinated forms as previously reported (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ).
      The requirement of p97/VCP and the Ufd1-Npl4 dimer in degradation of CD3δ were investigated in CD3δ/gp78 and CD3δ clones via RNA interference (RNAi)-mediated silencing of p97/VCP and Ufd1 expression. RNAi was first carried out in cells derived from CD3δ/gp78 clone-20, a randomly selected clone (Fig. 6, lanes 10-12). More than 90% reduction in the levels of p97/VCP and Ufd1 proteins was achieved by transfection with their specific siRNAs (Fig. 7a). p97/VCP knockdown leads to a dramatic stabilization of CD3δ (Fig. 7a, lanes 2-5), whereas inhibition of Ufd1 expression results in minimal effects (Fig. 7a, lanes 6 and 7). To confirm these results, we performed CHX chase analysis. As shown (Fig. 7b), degradation of CD3δ is largely diminished in p97/VCP knockdown cells (lanes 5-8 versus 1-4), whereas Ufd1 knockdown leads to stabilization of only a small amount of CD3δ that can be rapidly degraded (lanes 9-12 versus 1-4). Using a co-immunoprecipitation assay, we found that p97/VCP but not Ufd1 co-precipitates with gp78 in CD3δ/gp78 clone-20 cells (Fig. 7c, lane 4). However, when p97/VCP was immunoprecipitated from the same cells, Ufd1 was readily detected in precipitates (Fig. 7c, lane 5) supporting the notion that p97/VCP forms mutually exclusive complexes with either gp78 or Ufd1 in CD3δ/gp78 cells (Fig. 2c, lane 4).
      Figure thumbnail gr7
      FIGURE 7Effects of inhibiting p97/VCP or Ufd1 expression by RNAi on CD3δ degradation in CD3δ/gp78 clone-20 cells. a, inhibition of p97/VCP by siRNA stabilizes CD3δ, but Ufd1 siRNA has minimal effects in CD3δ/gp78 clone-20. Control siRNA (C) or siRNA for p97/VCP (V1 and V2 represent two different siRNAs targeting different regions of p97/VCP mRNA; shown in duplicate, lanes 2-5) or Ufd1 (U in duplicate, lanes 6 and 7) were transfected into CD3δ/gp78 clone-20 cells. Protein levels of CD3δ, p97/VCP, and Ufd1 were examined by immunoblotting. Actin was blotted as a loading control. b, inhibition of p97/VCP but not Ufd1 expression by RNAi markedly inhibits CD3δ degradation in CD3δ/gp78 clone-20 cells. CD3δ/gp78 clone-20 cells transfected as indicated were analyzed by CHX chase. RNAi and immunoblotting for HA, p97/VCP, and Ufd1 were performed as in a. Tubulin was blotted as loading control. c, gp78 is associated with p97/VCP but not Ufd1 in CD3δ/gp78 clone-20 cells. gp78 or p97/VCP was immunoprecipitated from CD3δ/gp78 clone-20 cells (lane 4 and 5). Anti-HA and anti-GFP antibodies were used as negative controls (lanes 2 and 3). Ufd1 in immunoprecipitates was detected by immunoblotting.
      To examine the requirement for Ufd1 in CD3δ degradation in CD3δ clone-5 cells, we silenced Ufd1 or p97/VCP in these cells. As in CD3δ/gp78 clone-20 cells, p97/VCP siRNAs markedly stabilizes CD3δ in CD3δ clone-5 cells (Fig. 8, a (lanes 3-6) and b (lanes 5-8)). Although Ufd1 siRNA has minimal effects on CD3δ degradation in CD3δ/gp78 clone-20 cells (Fig. 7, a (lanes 7 and 8) and b (lanes 9-12)), it significantly stabilizes CD3δ in CD3δ clone-5 cells (Fig. 8, a (lanes 7 and 8) and b (lanes 9-12)), suggesting that Ufd1 participates in CD3δ degradation in CD3δ clone-5 cells.
      Figure thumbnail gr8
      FIGURE 8Effects of inhibition of p97/VCP, Ufd1, or gp78 expression by RNAi on CD3δ degradation in CD3δ clone-5 cells. a, inhibition of p97/VCP or Ufd1 expression by RNAi stabilizes CD3δ in CD3δ clone-5 cells. b, inhibition of p97/VCP or Ufd1 expression by RNAi inhibits CD3δ degradation in CD3δ clone-5 cells. RNAi, CHX chase, and immunoblotting were performed as in , the effects of p97/VCP or Ufd1 knockdown on CD3δ stabilization were determined in 293 cells that express lower levels of CD3δ (lanes 1-3) and 293 cells that express higher levels of both CD3δ and gp78 (lanes 4-6). Equal amounts of total protein prepared from cells with indicated treatments were analyzed by immunoblotting for the specified proteins. Tubulin was blotted as a loading control. d, RNAi of endogenous gp78 inhibits CD3δ degradation. CD3δ clone-5 cells were transfected with siRNA expression empty vector pSUPER (C) or pSUPER-gp78siRNA1 (G) as previously reported (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). CD3δ degradation was monitored by CHX chase. Endogenous gp78 was detected by monoclonal anti-gp78 antibody 2G5. e, simultaneous knockdown of gp78 and Ufd1 produces synergistic inhibitory effects on CD3δ degradation. CD3δ clone-5 cells transfected with siRNA for Ufd1 (U) or gp78 (G) alone, or both (U + G) simultaneously were analyzed by CHX chase followed by immunoblotting for the specified proteins.
      We next asked whether the difference in the steady-state levels of CD3δ between CD3δ/gp78 clone-20 and CD3δ clone-5 cells affects the outcomes of RNAi for Ufd1 as we see in Figs. 7a and 8a. We therefore expressed lower levels of CD3δ in 293 cells with physiological levels of gp78 and higher levels of CD3δ in cells overexpressing gp78. Effects of knockdown of p97/VCP and Ufd1 on the levels of CD3δ in these cells were evaluated by immunoblotting. The results show that, although the steady-state levels of CD3δ are lower, knockdown of Ufd1 produces significant stabilization of CD3δ in cells with physiological levels of gp78 (Fig. 8c, lanes 1 and 3). Little effect on CD3δ levels is observed in cells that overexpress gp78 (Fig. 8c, lanes 4 and 6). However, knockdown of p97/VCP leads to marked stabilization of CD3δ in both cell types (Fig. 8c, lanes 2 and 5). These results are identical to those for CD3δ/gp78 clone-20 (Fig. 7a) and CD3δ clone-5 (Fig. 8a). Therefore, we conclude that any differences we observed in responses to Ufd1 knockdown in CD3δ/gp78 clone-20 and CD3δ clone-5 are not due to differences in their steady-state levels of CD3δ. Rather, the p97/VCP-Ufd1-Npl4 complex is involved in CD3δ degradation in CD3δ clone-5 cells, whereas Ufd1 is dispensable for CD3δ degradation in clone-20 cells that overexpress gp78. To determine if this Ufd1-independent degradation is true for other gp78 substrate, we examined the effects of p97/VCP and Ufd1 knockdown on the levels of the Z variant of α-1-antitrypsin, a newly identified gp78 substrate.
      Y. Shen, P. Ballar, and S. Fang, unpublished observation.
      The knockdown was carried out in 293 cells that stably express the Z variant of α-1-antitrypsin. The results showed that silencing p97/VCP but not Ufd1 stabilizes the Z variant of α-1-antitrypsin (supplemental Fig. S2), which further supports the possibility that gp78 mediates a Ufd1-independent ERAD pathway.
      To investigate whether gp78 is involved in CD3δ degradation in CD3δ clone-5 cells, gp78 expression was inhibited by RNAi, and degradation of CD3δ was evaluated by CHX chase. Our results show that silencing of gp78 expression significantly stabilizes CD3δ (Fig. 8d, lanes 5-8 versus 1-4), indicating that gp78 facilitates CD3δ degradation in CD3δ clone-5 cells.
      We then examined the Ufd1 dependence of the gp78-mediated ERAD in CD3δ clone-5 cells. The Ufd1 dependence was determined by comparing the stabilization effects on CD3δ in gp78 or Ufd1 single knockdown and gp78-Ufd1 double knockdown cells. If gp78 ubiquitinates CD3δ, and then the p97/VCP-Ufd1-Npl4 complex retrotranslocates the ubiquitinated CD3δ from the ER for degradation, we would expect little or no additive inhibitory effects on CD3δ degradation by a double knockdown of gp78 and Ufd1. However, the results from our CHX chase experiments show that the double knockdown of Ufd1 and gp78 produces a significant additive effect in the inhibition of CD3δ degradation (Fig. 8e, lanes 9-12). In addition, individual knockdown of gp78 or Ufd1 resulted in similar inhibitory effects (Fig. 8e, lanes 1-4 versus 5-8) suggesting that both gp78- and Ufd1-mediated pathways may contribute significantly to CD3δ degradation in CD3δ clone-5 cells. Interestingly, knockdown of Ufd1 appears to stabilize gp78 (Fig. 8e, lanes 1-4). This stabilization effect may represent a compensatory mechanism to ensure ERAD in the shortage of Ufd1.
      To assess the involvement of p97/VCP and Ufd1 in CD3δ degradation in a more quantitative way, we utilized pulse-chase analysis of CD3δ degradation in clone-5 and clone-20 cells. Consistent with the results obtained by the CHX chase analysis, knockdown of p97/VCP markedly stabilized CD3δ in both cell lines (Fig. 9a, lanes 5-8 versus 1-4). Although knockdown of Ufd1 had little effect on CD3δ degradation in CD3δ/gp78 clone-20 cells (Fig. 9a, lanes 1-4 versus 9-12, lower panel), it significantly stabilizes CD3δ in CD3δ clone-5 cells (Fig. 9a, lanes 1-4 versus 9-12, upper panel). Results were confirmed by densitometry quantification of three independent experiments (Fig. 9, b and c). These data strongly suggest that gp78 facilitates CD3δ degradation in an Ufd1-independent manner. In addition, the results help explaining why the steady-state levels of CD3δ are lower in clone-16 and -20 cells (Fig. 6). Collectively, our results suggest the presence of at least two pathways for CD3δ degradation in cells with physiological levels of ERAD machinery: the p97/VCP-Ufd1-Npl4 complex-mediated pathway and the gp78-mediated and Ufd1-independent pathway.
      Figure thumbnail gr9
      FIGURE 9Effects of p97/VCP and Ufd1 knockdown on CD3δ degradation determined by pulse-chase analysis. a, CD3δ clone-5 and CD3δ/gp78 clone-20 cells with knockdown of p97/VCP or Ufd1 were analyzed by pulse-chase. C: control siRNA; V1: VCP siRNA1; U: Ufd1 siRNA. b and c, densitometry quantification of CD3δ degradation from three independent pulse-chase experiments. The data were analyzed by ImageQuaNT and was expressed as mean ± S.D.

      DISCUSSION

      Studies of both yeast and higher eukaryotes have indicated that the Ufd1-Npl4 dimer is indispensable for CDC48/p97/VCP-mediated retrotranslocation of ubiquitinated ER proteins (
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Bays N.W.
      • Hampton R.Y.
      ). Here, we provide evidence to suggest a novel mechanism by which gp78-mediated ubiquitination is coupled with p97/VCP-mediated retrotranslocation without requiring Ufd1 (see a diagrammatic illustration of this model in Fig. 10). The molecular mechanism underlying this Ufd1-independent pathway is mediated through a novel p97/VCP-interacting motif, i.e. VIM of gp78. The VIM of gp78 interacts with the ND1 domain of p97/VCP, which is also the binding site for Ufd1 (
      • Meyer H.H.
      • Wang Y.
      • Warren G.
      ,
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ). Sharing of the interaction site leads gp78 and Ufd1 to form mutually exclusive complexes with p97/VCP (Fig. 4d). Therefore, gp78 physically excludes Ufd1 from the gp78-p97/VCP complex, thereby functionally excluding Ufd1 from gp78-mediated ERAD. However, our data does not exclude the possibility that other p97/VCP cofactor(s) might be involved in gp78-p97/VCP-mediated ERAD (Fig. 10).
      Figure thumbnail gr10
      FIGURE 10A model of gp78-mediated Ufd1-independent ERAD pathway. 1, the VIM of gp78 interacts with p97/VCP and recruits p97/VCP from the cytosol to the ER. 2, Ufd1 and Npl4 dimer is dissociated from p97/VCP as a result of gp78 interaction. The dissociated Ufd1-Npl4 dimer is not involved in gp78-p97/VCP-mediated ERAD. It is likely that another unknown cofactor of p97/VCP (labeled with “?”) may be required for this Ufd1-independent ERAD. 3, gp78, p97/VCP and possibly other factors couple ubiquitination with retrotranslocation leading to substrate degradation.
      The Ufd1-independent degradation of CD3δ becomes dominant in cells that overexpress gp78 as demonstrated by our RNAi experiments in CD3δ/gp78 clone-20 cells. Our data also support the presence of this pathway in cells that express endogenous levels of gp78. First, knockdown of gp78 stabilizes CD3δ in CD3δ clone-5 cells, indicating that endogenous gp78 is involved in ERAD (Fig. 8c). Second, although knockdown of Ufd1 accumulates CD3δ, CD3δ degradation still occurs although at a slower rate (Figs. 8b, 9a, and 9b). This suggests that an Ufd1-independent pathway exists in CD3δ clone-5 cells. Third, simultaneous knockdown of gp78 and Ufd1 results in a significant additive effect on CD3δ stabilization in CD3δ clone-5 cells (Fig. 8d). When expression of both gp78 and Ufd1 are almost abolished (>90% reduction), this additive inhibitory effect would only be possible if gp78 and Ufd1 act in parallel independent pathways. If gp78 and Ufd1 work sequentially, that is, gp78 mediates ubiquitination of CD3δ and then Ufd1 in conjunction with p97/VCP and Npl4 retrotranslocates the ubiquitinated CD3δ for degradation, knockdown of Ufd1 would have little effect when the function of gp78 is diminished. If gp78 and Ufd1 work in parallel in different degradation pathways, that is, they act in independent pathways on CD3δ degradation, simultaneous knockdown would block both pathways and produce significant additive effects of inhibition, which is what we have demonstrated. In contrast, p97/VCP is essential to both gp78 and Ufd1-mediated pathways. p97/VCP knockdown significantly stabilizes CD3δ regardless the levels of gp78 expression (Figs. 7, 8a, 8b, 9a, 9b, and 9c). This suggests that both Ufd1-dependent and -independent pathways require the function of p97/VCP.
      Studies have suggested that the p97/VCP-Ufd1-Npl4 complex is recruited to the ER by an ER-localized multiprotein complex through multiple interactions (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Schulze A.
      • Standera S.
      • Buerger E.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Koning F.
      • Kloetzel P.M.
      • Seeger M.
      ,
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ,
      • Lilley B.N.
      • Ploegh H.L.
      ) (Fig. 1b). In addition, other cytosolic ERAD components are also recruited to the ER by the multiprotein complex. For examples, both Derlin-1 and p97/VCP interact with the peptide:N-glycanase, a key enzyme that removes N-glycan from misfolded ER proteins before proteasomal degradation (
      • Li G.
      • Zhou X.
      • Zhao G.
      • Schindelin H.
      • Lennarz W.J.
      ,
      • Katiyar S.
      • Joshi S.
      • Lennarz W.J.
      ). Peptide:N-glycanase also interacts with hHR23B, a polyubiquitin-binding protein that chaperones polyubiquitinated ERAD substrates to the proteasomes for degradation (
      • Li G.
      • Zhou X.
      • Zhao G.
      • Schindelin H.
      • Lennarz W.J.
      ,
      • Lee J.H.
      • Choi J.M.
      • Lee C.
      • Yi K.J.
      • Cho Y.
      ). HERP, an ER stress-inducible protein, has recently been identified as a new member of the ERAD complex (
      • Schulze A.
      • Standera S.
      • Buerger E.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Koning F.
      • Kloetzel P.M.
      • Seeger M.
      ). However, how HERP regulates ERAD remains unknown. Nevertheless, there appears to be a large protein complex in the ER, which integrates recruitment, ubiquitination, retrotranslocation, deglycosylation, and proteasomal targeting of misfolded ER substrates during ERAD. The functional details and the dynamics of the complex are yet to be determined.
      This report and recent studies by others suggest that gp78 may be the central player in another ERAD pathway. gp78 acts not only as an ERAD E3, but also as a scaffold protein to assemble a complex that couples ubiquitination, retrotranslocation, and deglycosylation. gp78 has a multifunctional cytosolic tail that conveys E3 activity, binds polyubiquitin, and recruits Ubc7 and p97/VCP (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ,
      • Chen B.
      • Mariano J.
      • Tsai Y.C.
      • Chan A.H.
      • Cohen M.
      • Weissman A.M.
      ). Recent evidence indicates that p97/VCP recruited by gp78 is associated with peptide:N-glycanase (
      • Li G.
      • Zhou X.
      • Zhao G.
      • Schindelin H.
      • Lennarz W.J.
      ,
      • Chavan M.
      • Chen Z.
      • Li G.
      • Schindelin H.
      • Lennarz W.J.
      • Li H.
      ). We have now demonstrated that gp78 has a p97/VIM responsible for recruiting p97/VCP to the ER. Because gp78 and Ufd1 compete for binding to p97/VCP, gp78 recruits only p97/VCP but not the p97/VCP-Ufd1-Npl4 complex to the ER. Consistent with the absence of Ufd1 in the gp78-p97/VCP complex, our evidence also suggests that Ufd1 is dispensable in gp78-mediated CD3δ degradation. Other p97/VCP cofactor(s), however, may be involved in gp78-mediated ERAD (Fig. 10).
      Ufd1-independent ERAD may not be limited to gp78. VIM is also present in SVIP, another ER-membrane localized p97/VCP-binding protein (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ).
      P. Ballar and S. Fang, unpublished observation.
      SVIP, p47, and Ufd1 form mutually exclusive complexes with p97/VCP (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). It is likely that the VIM of SVIP interferes with the binding of p47 or Ufd1 to p97/VCP, as does the VIM of gp78. Therefore, SVIP may cooperate with other ERAD E3 to mediate Ufd1-independent ERAD. Thus, Ufd1-independent ERAD could represent one of the major pathways for disposal of misfolded ER proteins. In addition, many additional CDC48/p97/VCP cofactors with ubiquitin-binding capability have been identified (
      • Elsasser S.
      • Finley D.
      ,
      • Mullally J.E.
      • Chernova T.
      • Wilkinson K.D.
      ). Once the functions of the cofactors are fully uncovered, other mechanisms of coupling ubiquitination with retrotranslocation and degradation may be discovered. The presence of multiple ERAD pathways may be required to degrade different substrates and to facilitate efficient removal of misfolded proteins under different circumstances.

      Acknowledgments

      We thank Drs. Chou-Chi Li, Jun Yotoka, Hemmo Meyer, and Richard N. Sifers for reagents and Pamela Wright and Drs. Bruce Vogel, Sandra Honda, Mervyn J. Monteiro, and Bret Hassel for critical reading and comments on the manuscript.

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