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Identification of SVIP as an Endogenous Inhibitor of Endoplasmic Reticulum-associated Degradation

Open AccessPublished:September 14, 2007DOI:https://doi.org/10.1074/jbc.M704446200
      Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3δ from association with gp78 and p97/VCP, which is accompanied by decreases in CD3δ ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3δ and misfolded Z variant of α-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.
      Degradation of misfolded or unassembled proteins from the endoplasmic reticulum (ER),
      The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; E3, ubiquitin-protein isopeptide ligase; VIM, VCP-interacting motif; siRNA, short interfering RNA; GST, glutathione S-transferase; HA, hemagglutinin; IB, immunoblot; IP, immunoprecipitation; 2-OHM, 2-hydroxymyristic acid; CHX, cycloheximide.
      3The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; E3, ubiquitin-protein isopeptide ligase; VIM, VCP-interacting motif; siRNA, short interfering RNA; GST, glutathione S-transferase; HA, hemagglutinin; IB, immunoblot; IP, immunoprecipitation; 2-OHM, 2-hydroxymyristic acid; CHX, cycloheximide.
      a process known as ERAD, is essential for maintaining the homeostasis of the secretory pathway (
      • Hampton R.Y.
      ). Otherwise, delivery of misfolded proteins to their functional destinations or accumulation of misfolded proteins in the ER would have detrimental effects on cells (
      • Hampton R.Y.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ). In addition to its safeguard role, ERAD can directly regulate physiological processes that occur in the ER, for example, sterol-regulated degradation of HMG-CoA reductase, a rate-limiting enzyme in cholesterol synthesis, and inositol 1,4,5-trisphosphate (IP(3))-regulated degradation of IP(3) receptor that gates intracellular calcium release (
      • Hampton R.Y.
      ,
      • Song B.L.
      • Sever N.
      • DeBose-Boyd R.A.
      ,
      • Goldstein J.L.
      • DeBose-Boyd R.A.
      • Brown M.S.
      ,
      • Alzayady K.J.
      • Panning M.M.
      • Kelley G.G.
      • Wojcikiewicz R.J.
      ). ERAD is also known to control the levels of normal serum and glucocorticoid-induced kinase 1 and Cox-2 proteins (
      • Arteaga M.F.
      • Wang L.
      • Ravid T.
      • Hochstrasser M.
      • Canessa C.M.
      ,
      • Mbonye U.R.
      • Wada M.
      • Rieke C.J.
      • Tang H.Y.
      • Dewitt D.L.
      • Smith W.L.
      ). Furthermore, ERAD substrates have been extended to cytosolic and nuclear normal and/or misfolded proteins. For instance, ERAD ubiquitin ligase (E3) Hrd1 interacts with cytosolic p53 and misfolded Huntingtin protein and targets them to the proteasomes for degradation (
      • Yamasaki S.
      • Yagishita N.
      • Sasaki T.
      • Nakazawa M.
      • Kato Y.
      • Yamadera T.
      • Bae E.
      • Toriyama S.
      • Ikeda R.
      • Zhang L.
      • Fujitani K.
      • Yoo E.
      • Tsuchimochi K.
      • Ohta T.
      • Araya N.
      • Fujita H.
      • Aratani S.
      • Eguchi K.
      • Komiya S.
      • Maruyama I.
      • Higashi N.
      • Sato M.
      • Senoo H.
      • Ochi T.
      • Yokoyama S.
      • Amano T.
      • Kim J.
      • Gay S.
      • Fukamizu A.
      • Nishioka K.
      • Tanaka K.
      • Nakajima T.
      ,
      • Yang H.
      • Zhong X.
      • Ballar P.
      • Luo S.
      • Shen Y.
      • Rubinsztein D.C.
      • Monteiro M.J.
      • Fang S.
      ). Saccharomyces cerevisiae ERAD E3 Doa10 is spatially sorted to the inner nuclear membrane and targets nuclear-localized substrates for degradation (
      • Deng M.
      • Hochstrasser M.
      ).
      Degradation of ER-localized misfolded proteins occurs in the cytosol by the proteasomes. Thus, luminal proteins have to be transported across the membrane, and membrane-integrated proteins have to be dislodged from the membrane to reach the proteasomes. These processes are collectively called retrotranslocation (
      • Hampton R.Y.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ). Increasing evidence indicates that actions of large protein complexes in the ER integrate the processes of misfolded protein selection, retrotranslocation, ubiquitination, and degradation during ERAD (
      • Hampton R.Y.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ). Indispensable to these complexes are the ER membrane-spanning RING finger E3s for ubiquitination and the AAA ATPase CDC48/p97/VCP for retrotranslocation (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ). This is best characterized in S. cerevisiae in which distinct E3 complexes define different ERAD pathways (
      • Carvalho P.
      • Goder V.
      • Rapoport T.A.
      ,
      • Denic V.
      • Quan E.M.
      • Weissman J.S.
      ). Proteins with misfolded ER-luminal domains are targeted to the ERAD-L pathway. In this pathway, the Hrd1p/Hrd3p ligase forms a core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p and Kar2p, forming a luminal protein surveillance complex. Substrates with misfolded intramembrane domains are targeted to the ERAD-M pathway, which differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p E3. All three pathways converge at the Cdc48p ATPase complex. The general scheme of the S. cerevisiae ERAD pathways is applicable to that in mammalian cells. So far, five ER membrane-spanning RING finger E3s, including gp78, Hrd1, TEB4, Rma1, and RFP2, have been identified in mammalian ERAD (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Kikkert M.
      • Doolman R.
      • Dai M.
      • Avner R.
      • Hassink G.
      • van Voorden S.
      • Thanedar S.
      • Roitelman J.
      • Chau V.
      • Wiertz E.
      ,
      • Hassink G.
      • Kikkert M.
      • Voorden S.
      • Lee S.J.
      • Spaapen R.
      • Laar T.
      • Coleman C.S.
      • Bartee E.
      • Fruh K.
      • Chau V.
      • Wiertz E.
      ,
      • Younger J.M.
      • Chen L.
      • Ren H.Y.
      • Rosser M.F.
      • Turnbull E.L.
      • Fan C.Y.
      • Patterson C.
      • Cyr D.M.
      ,
      • Lerner M.
      • Corcoran M.
      • Cepeda D.
      • Nielsen M.L.
      • Zubarev R.
      • Ponten F.
      • Uhlen M.
      • Hober S.
      • Grander D.
      • Sangfelt O.
      ). Other components of S. cerevisiae ERAD pathways have also been found in mammalian cells, such as p97/VCP (homologue of CDC48), Derlin1, Derlin2, and Derlin3 (homologues of Der1p), and Sel1L (homologue of Hrd3p) (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Lilley B.N.
      • Ploegh H.L.
      ,
      • Oda Y.
      • Okada T.
      • Yoshida H.
      • Kaufman R.J.
      • Nagata K.
      • Mori K.
      ,
      • Hampton R.Y.
      • Gardner R.G.
      • Rine J.
      ,
      • Mueller B.
      • Lilley B.N.
      • Ploegh H.L.
      ). Unlike in S. cerevisiae, the ERAD pathways in mammalian cells are largely undefined. However, a link between ERAD E3s and p97/VCP ATPase has been demonstrated in mammalian cells and is essential for mammalian ERAD (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Lilley B.N.
      • Ploegh H.L.
      ,
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ).
      We have recently reported that gp78 directly interacts with p97/VCP through a newly identified p97/VCP-interacting motif (VIM) (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ). This interaction is essential for degradation of ERAD substrates CD3δ and the Z variant of α-1-antitrypsin (ATZ). The putative retrotranslocation channel Derlin1 has been shown to be part of the gp78 and p97/VCP complex (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). Interestingly, a highly conserved VIM is also found in the small p97/VCP-interacting protein (SVIP) (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ). SVIP was isolated by a yeast two-hybrid screen using p97/VCP as bait (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). Overexpression of SVIP causes vacuolization of cells, but its physiological role is unknown (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). The sharing of VIM between gp78 and SVIP prompted us to evaluate a possible regulatory role of SVIP on gp78-mediated ERAD. We found that SVIP forms a complex with Derlin1 and p97/VCP. We provided evidence that SVIP regulates the formation of gp78-p97/VCP-Derlin1 complex, which leads to a change in substrate association with gp78 and p97/VCP and, eventually, changes in the efficacy of ERAD. These findings suggest that SVIP is an endogenous inhibitor of ERAD, acting through the inhibition of the assembly of the gp78-p97/VCP-Derlin1 complex. Thus, SVIP may act to prevent excessive ERAD that may cause damage to cells.

      EXPERIMENTAL PROCEDURES

      Plasmids, siRNAs, Antibodies, and Cell Lines

      SVIP open reading frame was amplified by reverse transcription PCR from total RNA isolated from 293 cells. A His6 tag was added in-frame to the C terminus of SVIP during PCR amplification, and the PCR product was cloned into the mammalian expression vector pCIneo via EcoRI and SalI sites (pCIneo-SVIP). pCIneo-SVIP(G2A) was created by site-directed mutagenesis to mutate myristoylation site glycine 2 to alanine. pCIneo-SVIP-VIM mutant was generated by simultaneously mutating the conserved Arg-22 to Glu-, Leu-25 to Gln-, Ala-26 to Val, Arg-31 to Asp, and Arg-32 to Glu. pCI-HA-CD3δ and pGEX4T3-SVIP have been previously described (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). hATZ/pcDNA3.1Zeo(+) was a kind gift from Dr. Richard N. Sifers (
      • Wu Y.
      • Swulius M.T.
      • Moremen K.W.
      • Sifers R.N.
      ). pGEX-KG-Derlin1-Ccr (encoding GST-Drlc) was generously provided by Dr. Michael Seeger (
      • Schulze A.
      • Standera S.
      • Buerger E.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Koning F.
      • Kloetzel P.M.
      • Seeger M.
      ). pcDNA3.1-HA-Derlin1 was generously provided by Dr. Yihong Ye (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). pCMV-HA-gp78 (1–643, full-length) and pCMV-HA-gp78 (1–308, transmembrane domain) were kindly provided by Dr. Russell A. DeBose-Boyd (
      • Song B.L.
      • Sever N.
      • DeBose-Boyd R.A.
      ).
      Silencer© pre-designed siRNAs for negative control number 1, p97/VCP (sense sequence, GGGCACAUGUGAUUGUUAU), SVIP (sense sequence, GACAAAAAGAGGCUGCAUC) were purchased from Ambion. Polyclonal anti-SVIP antibodies were generated by immunizing rabbit with purified glutathione S-transferase (GST)-SVIP protein. Anti-GST antibodies were removed by passing the anti-GST-SVIP serum through GST-agarose beads. Monoclonal antibodies against hemagglutinin (HA) and actin were purchased from Sigma. Anti-polyubiquitin and anti-green fluorescent protein were acquired from Santa Cruz. Anti-p97/VCP and anti-calnexin antibodies were purchased from Affinity BioReagents. Anti-gp78 and anti-Hrd1 antibodies have been previously described (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ). Polyclonal anti-Derlin1 and anti-VIMP antibodies were kindly provided by Dr. Yihong Ye (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ). Anti-Npl4 antibodies were generated in rabbits using purified His6-Npl4. Rabbit polyclonal anti-human α-1-antitrypsin was purchased from Biomeda. Human horseradish peroxidase-conjugated anti-α-1-antitrypsin was acquired from Bethyl Laboratories.

      Stable Cell Lines

      293 cells that stably express HA-CD3δ alone (clone-5) and HA-CD3δ together with gp78 (clone-20) have been previously reported (
      • Arteaga M.F.
      • Wang L.
      • Ravid T.
      • Hochstrasser M.
      • Canessa C.M.
      ). To generate cell lines that stably express ATZ, plasmid encoding hATZ was co-transfected with pBABE vector that confers puromycin resistance. 24 h after the transfection, 2.5 μg/ml puromycin was added to eliminate non-transfected cells. Positive clones were characterized by immunoblotting.

      Immunoblotting (IB) and Immunoprecipitation (IP)

      293 cells were seeded at 2.5 × 105/well in 6-well plates or 1.2 × 106/100-mm dish prior to the day of transfection with Lipofectamine 2000 (Invitrogen) (for plasmids and siRNAs) or by calcium phosphate precipitation (for plasmids only). Cells transfected with plasmids were collected 24 h after transfection, whereas those transfected with siRNA were harvested 72 h post-transfection. IB and IP were performed as we previously described (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ).

      Subcellular Fractionation

      Alkaline Extraction—Microsomes were isolated as described (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ) and then incubated with 0.1 m Na2CO3, pH 11, for 20 min at room temperature. After the incubation, microsomes were pelleted by centrifugation at 105,000 × g for 10 min at 4 °C. The resulting microsomes and supernatants were processed for IB.
      Proteinase K Digestion Assay—Microsomes were incubated with 0, 3.1, 6.25, 12.5, 25, 50, 100 μg/ml proteinase K in 1× phosphate-buffered saline for 30 min at room temperature before being processed for IB.
      N-Myristoylation Inhibition—1 mm 2-OHM (2-hydroxymyristic acid) (Sigma) was delivered to 293 cells in a complex with fatty acid-free bovine serum albumin (Sigma) as previously described (
      • Nadler M.J.
      • Harrison M.L.
      • Ashendel C.L.
      • Cassady J.M.
      • Geahlen R.L.
      ), and cells were incubated for 24 h. Because unmyristoylated cytosolic SVIP is not very stable, cells were treated with proteasome inhibitor for 5 h before being processed for fractionation and IB.
      Gradient Fractionation—293 cells were homogenized in buffer B (0.25 m sucrose, 1 mm EDTA, 10 mm HEPES-NaOH, pH 7.4) and then centrifuged at 3000 × g for 10 min to remove nuclei and unbroken cells. The post-nuclear supernatant was layered on top of a preformed 0–25% iodixanol gradient in buffer B and centrifuged at 200,000 × g for 2.5 h, after which fractions were collected from the bottom of the tube. Equal volume of each fraction was processed for IB.

      RESULTS

      SVIP Is a Membrane-anchored Protein—SVIP has been reported to be a membrane-associated protein, but it possesses neither a transmembrane domain nor a signal peptide (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). How it associates with the membrane is not clear. To address this issue, we carried out alkaline extraction of the microsomes isolated from 293 cells. We found that Na2CO3 has no effect on SVIP-membrane association, as seen on the membrane-spanning protein Derlin1 (Fig. 1A). As a positive control, a significant amount of p97/VCP that is known to be peripherally associated with the microsomes is released into the cytosol (Fig. 1A, lane 2 versus 4). This result is consistent with the previous report that SVIP may be attached to membrane through myristoylation (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). To test this possibility, we mutated the putative myristoylation site glycine-2 to alanine and generated a mutant SVIP (SVIP(G2A)) that is defective in myristoylation. We found that SVIP(G2A) is exclusively localized in the cytosol (Fig. 1B, lanes 3, 4 versus 5, 6). The difference is not due to overexpression, since SVIP(G2A) was still in the cytosol when its expression was markedly reduced (lanes 7, 8 versus 9, 10). Furthermore, the membrane anchorage of SVIP can be abolished by the N-myristoylation inhibitor 2-hydroxymyristic acid (2-OHM). Treatment with 2-OHM slightly increased the mobility of SVIP and resulted in its cytosolic localization (Fig. 1C), which is consistent with inhibition of SVIP myristoylation. Thus, it is likely that SVIP is membrane-anchored through myristoylation on glycine 2. In agreement, we demonstrated that SVIP is attached on the cytosolic surface of microsomes, since proteinase K completely digested SVIP, while the intraluminal domain of calnexin remained protected by the microsomal membrane (Fig. 1D).
      Figure thumbnail gr1
      FIGURE 1SVIP is anchored to microsomal membrane, probably through myristoylation. A, alkaline extraction does not affect SVIP-microsome association. 293 cells were homogenized and the microsomes (m) were isolated from the post-nuclear homogenate as we previously described (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ). For alkaline extraction, the microsomes were incubated in 0.1 m Na2CO3, pH 11, or in 1× phosphate-buffered saline as a control. After the incubation, the microsomes were pelleted, and the supernatants (s) were collected. These fractions were analyzed by IB using anti-SVIP, anti-Derlin1, and anti-p97/VCP antibodies. p97/VCP and Derlin1 were used as controls for peripheral protein and ER membrane-anchored protein, respectively. B, wild-type (Wt)-SVIP, but not myristoylation-deficient mutant SVIP(G2A), localizes to the microsomes. 293 cells transfected either with pCIneo, Wt-SVIP, or SVIP(G2A) were processed for fractionation into microsomes (m) and cytosol (c). The localization of Wt-SVIP and SVIP-G2A was determined by blotting with anti-His6 antibody to detect their His6 tags. C, 2-hydroxymyristic acid (2-OHM) inhibits endogenous SVIP anchorage to membrane. 293 cells were treated with 2-OHM or its vehicle (ethanol: EtOH) followed by fractionation into microsomes (m) and cytosol (c). D, proteinase K digestion assay used to determine the membrane topology of SVIP. Microsomes prepared from 293 cells were incubated with increasing amounts of proteinase K (0–100 μg/ml) for 30 min before processing for IB with anti-SVIP and anti-calnexin (Cnx) antibodies.
      SVIP Forms a Complex with p97/VCP and Derlin1—SVIP is localized to the ER and other membranes (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). To investigate whether the two known VIM-containing proteins, SVIP and gp78, are in the same membrane fraction, post-nuclear supernatants from 293 cells were fractionated on an iodixanol (Optiprep) gradient. Indeed, SVIP cofractionated with gp78 along with other ER membrane proteins such as Derlin1, Hrd1, and calnexin (Fig. 2A). As expected, a fraction of p97/VCP cofractionated with SVIP and gp78 (Fig. 2A).
      Figure thumbnail gr2
      FIGURE 2SVIP forms a complex with p97/VCP and Derlin1. A, SVIP is cofractionated with ERAD machineries. The post-nuclear homogenates of 293 cells were fractionated on a 0–25% iodixanol gradient. Distributions of endogenous SVIP, Hrd1, gp78, Derlin1, calnexin, Golgin-97, transferrin receptor (TfR), p97/VCP, Ufd1, and Npl4 were revealed by IB. B, SVIP forms a complex with Derlin1 and p97. Lysates prepared from 293 cells that were transfected with His6-SVIP were used to immunoprecipitate SVIP (lane 2) using anti-His6 antibody. Endogenous p97/VCP (lane 4) and VIMP (lane 5) were immunoprecipitated with anti-VCP and anti-VIMP, respectively. Anti-GFP was used as a negative control for IP. Lane 1 contains 1/10 of the input proteins used for IP. Immunoprecipitates were processed for blotting for the indicated proteins. C, silencing p97/VCP expression abolishes SVIP-Derlin1 interaction. His6-SVIP-expressing 293 cells were transfected either with control (C) or p97/VCP (V) siRNA. Three days after siRNA transfection, cells were lysed and processed for IP with anti-His6. SVIP-associated Derlin1 was revealed by IB with anti-Derlin1 antibody. D, schematic representation of SVIP-p97/VCP-Derlin1 complex.
      To determine whether SVIP interacts with ERAD components other than p97/VCP, we performed a co-immunoprecipitation assay. Among the additional five proteins examined, only Derlin1 strongly co-immunoprecipitated with SVIP (Fig. 2B, lane 2). As previously reported, VIMP interacts with p97/VCP, Derlin1, and gp78 (Fig. 2A, lane 5), whereas p97/VCP co-immunoprecipitated with SVIP, Derlin1, gp78, Hrd1, and its cofactors Ufd1-Npl4 heterodimer (lane 4). Thus, SVIP and gp78 not only share a common motif, VIM, but also have common interacting proteins, p97/VCP and Derlin1. Next, we assessed how the SVIP-p97/VCP-Derlin1 complex is formed by inhibiting p97/VCP expression with RNA interference. Silencing p97/VCP expression markedly reduced the SVIP-Derlin1 interaction (Fig. 2C, lanes 5, 6), suggesting that the trimeric complex is formed through simultaneous binding of SVIP and Derlin1 to p97/VCP (illustrated in Fig. 2D). The same results were obtained by a GST-SVIP pulldown assay (supplemental Fig. S1A, lanes 5, 6). Interestingly, p97/VCP also appears to be essential for the formation of the gp78-p97/VCP-Derlin1 complex as demonstrated by GST-gp78VIM and GST fusion of the Derlin1 C-terminal tail (GST-Drlc) pulldown assays (supplemental Fig. S1A, lanes 3, 4, and S1B, lanes 3, 4). In addition, the transmembrane domains of gp78 interact with Derlin1 (supplemental Fig. S2), suggesting that p97/VCP interactions enhance the binding of gp78 and Derlin1. Considering that the SVIP-p97/VCP-Derlin1 complex does not contain gp78 and that VIM is present in both gp78 and SVIP, it is likely that SVIP and gp78 form a mutually exclusive complex with p97/VCP-Derlin1.
      SVIP Regulates gp78-mediated ERAD—Overexpression of SVIP induces cellular vacuolation (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ), and electron microscopy revealed that the vacuoles represent dilated ER (
      • Nagahama M.
      • Suzuki M.
      • Hamada Y.
      • Hatsuzawa K.
      • Tani K.
      • Yamamoto A.
      • Tagaya M.
      ). We speculated that such vacuoles might be caused by accumulation of misfolded ER proteins and that SVIP might be an inhibitor of ERAD. To test this possibility, we determined the effects of SVIP on ERAD. Increasing amounts of SVIP were expressed in 293 cells that stably express CD3δ, a well known ERAD substrate (
      • Chen B.
      • Mariano J.
      • Tsai Y.C.
      • Chan A.H.
      • Cohen M.
      • Weissman A.M.
      ). We found that SVIP causes a dose-dependent accumulation of CD3δ (Fig. 3A). The accumulation was due to decreased degradation as shown by cycloheximide (CHX) chase analysis in 293 cells with approximately three times overexpression of SVIP (
      • Chen B.
      • Mariano J.
      • Tsai Y.C.
      • Chan A.H.
      • Cohen M.
      • Weissman A.M.
      ) (Fig. 3B). When a similar experiment was performed with the membrane anchorage mutant form of SVIP (SVIP(G2A)), the same result was achieved. Overexpression of SVIP(G2A) significantly inhibits CD3δ degradation (Fig. 3C), indicating that the membrane anchorage of SVIP is not required for ERAD inhibition. Consistently, SVIP(G2A) remains interacting with p97/VCP and Derlin1 (Fig. 3D, lane 6). As expected, mutation of the VIM of SVIP (SVIP(VIMm)) abrogates SVIP interaction with p97/VCP and Derlin1 (Fig. 3D, lane 5). The slower mobility of SVIP(VIMm) may be caused by amino acid substitutions and/or post-translational modification.
      Figure thumbnail gr3
      FIGURE 3SVIP inhibits ERAD. A, SVIP causes dose-dependent accumulation of CD3δ. 293 cells stably expressing HA-CD3δ (HA-CD3δ clone-5 cell line) (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ) were transfected with increasing amounts of pCIneo-SVIP (0, 0.5, 1, 2, 4 μg). The levels of CD3δ were determined by IB with anti-HA. Actin was blotted as loading control. B, SVIP inhibits CD3δ degradation. HA-CD3δ clone-5 cells were transfected either with pCIneo or pCIneo-SVIP (2 μg). 24 h after transfection, the cells were treated with 100 μg/ml CHX and chased for the indicated times. The levels of CD3δ were determined by IB. Densitometric analysis of HA-CD3δ levels is shown in the bottom panel (mean ± S.D., n = 3). C, SVIP(G2A) inhibits CD3δ degradation. HA-CD3δ clone-5 cells were transfected with pCIneo, pCIneo-SVIP (2 μg), or SVIP(G2A)(4 μg due to its stability). 24 h after the transfection, cells were analyzed by CHX chase as in panel B. D, Wt SVIP and SVIP(G2A), but not SVIP(VIMm), interact with p97/VCP and Derlin1. 293 cells transfected as indicated were processed for IP using anti-His6 antibody to precipitate His6-tagged SVIP and its mutants. Precipitates were processed for IB for the indicated proteins. E, overexpression of SVIP abolishes p97/VCP-CD3δ interaction. HA-CD3δ was immunoprecipitated from HA-CD3δ clone-5 cells transfected with pCIneo-SVIP or empty vector (2μg). CD3δ-associated p97/VCP and Derlin1 was determined by IB with anti-p97/VCP and -Derlin1 antibodies. F, SVIP inhibits gp78-p97/VCP interaction. 293 cells transfected as indicated were processed for IP for endogenous gp78 with anti-gp78 antibody. gp78-bound p97/VCP was revealed by blotting with anti-VCP antibody. G, SVIP inhibits gp78-CD3δ interaction and CD3δ ubiquitination. HA-CD3δ clone-5 cells transfected with pCIneo-SVIP (2μg) were treated with proteasome inhibitor lactacystin (10 μm) for 6 h to accumulate ubiquitinated HA-CD3δ before processing for IP for HA-CD3δ. Immunoprecipitates were processed for blotting for ubiquitin, HA-CD3δ, and gp78. * indicates IgG HC. H, SVIP inhibits Derlin1-gp78 interaction. HA-Derlin1 alone or along with SVIP (2 μg) was expressed in 293 cells. 24 h after the transfection, cells were processed for IP for HA-Derlin1. The associated gp78 was detected by IB.
      gp78 enhances CD3δ binding to p97/VCP and facilitates CD3δ degradation (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). The apparent opposite effect of SVIP on CD3δ degradation prompted us to determine the effect of SVIP on CD3δ-p97/VCP association. Consistent with the observed decreased degradation in the previous experiment, overexpression of SVIP inhibits p97/VCP binding to CD3δ (Fig. 3E) and gp78 (Fig. 3F). Furthermore, in SVIP-overexpressing cells, gp78 did not interact with CD3δ, which correlates with a significant decrease in the level of ubiquitinated CD3δ (Fig. 3G). To further confirm this result, we immunoprecipitated HA-CD3δ in denatured cell lysates. Consistently, SVIP overexpression significantly decreased the levels of ubiquitinated CD3δ (supplemental Fig. S3). In addition, SVIP overexpression diminishes gp78-Derlin1 interaction (Fig. 3H). Collectively, these experiments indicate that SVIP inhibits the interactions of gp78 with CD3δ, p97/VCP, and Derlin1, resulting in inhibition of CD3δ ubiquitination and subsequent loading to p97/VCP for retrotranslocation.
      Next, we asked whether the endogenous SVIP plays an inhibitory role in gp78-mediated ERAD. We again utilized 293 cells that stably express CD3δ. RNA interference was employed to silence endogenous SVIP expression. Degradation of CD3δ was assessed by CHX chase analysis. Inhibition of SVIP expression significantly increased CD3δ degradation (Fig. 4A). A similar experiment was performed on ATZ, a newly identified luminal substrate for gp78 (
      • Shen Y.
      • Ballar P.
      • Fang S.
      ). As predicted, silencing SVIP also decreased the intracellular levels of ATZ, which correlates with reduced ATZ secretion (Fig. 4B). These data further support the hypothesis that endogenous SVIP negatively regulates the function of gp78s. To determine whether endogenous SVIP inhibits gp78 interaction with p97/VCP and Derlin1, we silenced the expression of SVIP and evaluated changes in the formation of gp78-p97/VCP-Derlin1 complex by co-immunoprecipitation. Inhibition of SVIP expression markedly augmented the association of p97/VCP and Derlin1 with gp78, consistent with the fact that silencing SVIP enhances ERAD. To further substantiate gp78 as a target inhibited by SVIP, we studied the effects of SVIP silencing on CD3δ degradation in 293 cells that overexpress both gp78 and CD3δ. We predicted that overexpression of gp78 will overcome the inhibitory effect exerted by endogenous SVIP and that silencing SVIP would have no effect on CD3δ degradation in cells overexpressing gp78. This was indeed the case (Fig. 4D). Collectively, these results suggest that SVIP is an endogenous inhibitor for gp78-mediated ERAD that acts through its common motif shared with gp78 and uncouples gp78 from its substrates, p97/VCP and Derlin1. Changes in the levels of SVIP and gp78 proteins appear to control the efficacy of ERAD (Fig. 3, A and B, and Fig. 4, A, B, and D). The next question is whether the expressions of SVIP and gp78 proteins are modulated under ER stress, a condition known to regulate the efficacy of ERAD. Interestingly, tunicamycin-induced ER stress inversely regulated the levels of SVIP and gp78 proteins in a time-dependent manner, which correlates well with previous reports that ER stress enhances, but prolonged ER stress (17 h) inhibits, ERAD (
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Doong H.
      • Fang S.
      ,
      • Menendez-Benito V.
      • Verhoef L.G.
      • Masucci M.G.
      • Dantuma N.P.
      ).
      Figure thumbnail gr4
      FIGURE 4Endogenous SVIP inhibits CD3δ degradation. A, silencing SVIP expression increases CD3δ degradation. HA-CD3δ clone-5 cells were transfected with control (C) or SVIP (S) siRNA. Three days after transfection, degradation of HA-CD3δ was analyzed by CHX chase. SVIP was blotted with polyclonal anti-SVIP antibodies. Densitometric analysis of HA-Cd3δ levels by ImageQuant is shown in the lower panel (mean ± S.D., n = 3). B, silencing SVIP expression reduces the levels of intracellular ATZ accompanied by decreased ATZ secretion. 293 cells stably expressing ATZ were transfected with control (C) or SVIP (S) siRNA. Three days after transfection, CHX chase was performed. Intracellular level of ATZ (i) was detected by IB with horseradish peroxidase-conjugated anti-ATZ antibody. To determine the secreted ATZ (s), media were processed for IP with anti-ATZ antibody. Actin was blotted as loading control. * indicates IgG HC in lanes 4, 5, 9, 10. C, silencing endogenous SVIP expression enhances the formation of gp78-p97/VCP-Derlin1 complex. Control (C) or SVIP (S) siRNA was transfected into 293 cells. Three days after transfection, cells were processed for IP for gp78. Co-immunoprecipitated Derlin1 and p97/VCP were detected by IB with anti-Derlin1 and anti-VCP, respectively. SVIP was blotted to determine knock down efficiency. D, gp78 overexpression diminishes the inhibitory effects of SVIP on CD3δ degradation. 293 cells that stably express both gp78 and CD3δ (gp78-CD3δ clone-20 cells) (
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ) were transfected as the indicated siRNA. Three days after transfection, cells were used for CHX chase analysis. Densitometric analysis of HA-CD3δ levels is shown in the lower panel (mean ± S.D., n = 3). E, tunicamycin treatment inversely regulates the levels of SVIP and gp78 proteins. 293 cells were treated with tunicamycin (2.5 μg/ml) for the indicated time and then processed for IB. The relative expression levels of SVIP and gp78 proteins were quantified by densitometry and expressed in the graph.

      DISCUSSION

      The central components of the ERAD pathways include the ER resident E3s and the cytosolic CDC48/p97/VCP ATPase (
      • Carvalho P.
      • Goder V.
      • Rapoport T.A.
      ,
      • Denic V.
      • Quan E.M.
      • Weissman J.S.
      ). Multiple ER membrane-anchored proteins interact with and link CDC48/p97/VCP to ERAD E3s, thereby promoting association of ubiquitinated ER proteins with the ATPase for retrotranslocation (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Lilley B.N.
      • Ploegh H.L.
      ). In this study, we found that myristoylation targets SVIP to the ER membrane where it competes with gp78 to bind to p97/VCP and Derlin1. As a result, SVIP reduces the association of ERAD substrates with gp78 and p97/VCP, thereby inhibiting substrate ubiquitination and subsequent ERAD steps. This study identifies SVIP as the first endogenous inhibitor of ERAD that uses a novel mechanism through inhibiting the assembly of the gp78-p97/VCP-Derlin1 complex.
      Previous studies have focused on the mechanisms of ERAD and how ERAD is enhanced under ER stress (
      • Hampton R.Y.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ,
      • Travers K.J.
      • Patil C.K.
      • Wodicka L.
      • Lockhart D.J.
      • Weissman J.S.
      • Walter P.
      ). However, the control mechanism of ERAD activity is largely unknown. As a general mechanism, activated cellular events have to be turned off after having fulfilled their tasks, such as in the cases of the activated receptor tyrosine kinases and transcription factors (
      • Haglund K.
      • Sigismund S.
      • Polo S.
      • Szymkiewicz I.
      • Di Fiore P.P.
      • Dikic I.
      ,
      • Muratani M.
      • Tansey W.P.
      ). Failure to control the durations of receptor tyrosine kinase signaling and gene transcription can cause devastating diseases. Therefore, it is conceivable that activated ERAD in response to accumulation of misfolded proteins in the ER has to be controlled once the misfolded proteins have been removed. A similar mechanism by which ERAD is controlled may be through degradation of key components of the ERAD machineries. For example, gp78 itself is degraded by ERAD (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ). Importantly, gp78 degradation is inhibited under ER stress, which correlates with an increase in ERAD (
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). Thus, when misfolded proteins are accumulated in the ER, gp78 is stabilized and it aids to remove misfolded proteins. Once the accumulated proteins in the ER have been eliminated, gp78 is degraded, thereby preventing excessive ERAD that may cause damage to cells. The present finding on the regulation of the assembly of ERAD machinery by SVIP represents another mechanism of control for ERAD activity.
      Although p97/VCP binds gp78 and SVIP in a mutually exclusive manner, both p97/VCP-gp78 and p97/VCP-SVIP can associate with Derlin1. This type of interaction is reminiscent of the Hrd1-p97/VCP-Derlin1 complex, in which p97/VCP simultaneously binds Derlin1 and Hrd1 (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ). Our study also revealed an interaction between the transmembrane domains of gp78 and Derlin1, supporting the possibility that gp78 and Derlin1 may be part of the protein retrotranslocation channel as previously suggested (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ). Derlin1 has two homologues, Derlin2 and Derlin3. Derlin2 also forms complex with p97/VCP and Hrd1 (
      • Lilley B.N.
      • Ploegh H.L.
      ). It would be important to know whether SVIP also interacts with Derlin2 and Derlin3. If it does, SVIP may play a more general inhibitory role on ERAD. CDC48/p97/VCP ATPase is the converging point of probably all the characterized ERAD pathways. Inhibition of this ATPase is expected to disrupt the ERAD process. The question is why SVIP also uncouples Derlin1 from gp78. Derlin1 has been proposed as the long sought after channel for retrotranslocation (
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ). Thus, simultaneous sequestration of p97/VCP and Derlin1 by SVIP would inhibit the targeting of misfolded proteins to the retrotranslocation channel and subsequent retrotranslocation. Additionally, SVIP diminished the gp78-CD3δ interaction, which results in stabilization of non-ubiquitinated CD3δ. By doing so, SVIP may enhance protein trafficking through the ER/Golgi. In support of this possibility, we found that silencing SVIP expression increases ATZ degradation and decreases ATZ secretion (Fig. 4B).
      Our data presented strongly suggest that p97/VCP and Derlin1 can form a complex with either gp78 or SVIP. The gp78 complex facilitates ERAD, whereas the SVIP complex inhibits ERAD; the stoichiometry between these two complexes dictates the efficacy of gp78-mediated ERAD (Fig. 5). Importantly, the stoichiometry is apparently regulated under ER stress (Fig. 4E).
      Figure thumbnail gr5
      FIGURE 5A model illustrates the regulation of gp78-mediated ERAD by SVIP. SVIP and gp78 form mutually exclusive complexes with p97/VCP and Derlin1. When the complex shifts to gp78, ERAD is facilitated. Otherwise, ERAD is inhibited. VIM, p97/VCP-interacting motif of gp78.

      Acknowledgments

      We thank Dr. Yihong Ye for providing antibodies and plasmids and Drs. Russell A. DeBose-Boyd, Richard N. Sifers, and Michael Seeger for providing plasmid constructs. We thank Drs. Martin F. Flajnik and Robert Cohen for critical review of the manuscript.

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