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

Petek Ballar,Masami Nagahama,Yuxian Shen,Yongwang Zhong,Shengyun Fang,Mitsuo Tagaya

doi:10.1074/jbc.m704446200

Abstract

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 CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-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.

Highlights

Tional destinations or accumulation of misfolded proteins in the endoplasmic reticulum (ER) would have detrimental effects on cells [1, 2]
We found that myristoylation targets small p97/VCP-interacting protein (SVIP) to the ER membrane where it competes with gp78 to bind to p97/VCP and
This study identifies SVIP as the first endogenous inhibitor of ER-associated degradation (ERAD) that uses a novel mechanism through inhibiting the assembly of the gp78-p97/VCP-Derlin1 complex

Summary

EXPERIMENTAL PROCEDURES

SVIP open reading frame was amplified by reverse transcription PCR from total RNA isolated from 293 cells. 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-CD3␦ and pGEX4T3-SVIP have been previously described [15, 27]. PGEX-KG-Derlin1-Ccr (encoding GST-Drlc) was generously provided by Dr Michael Seeger [29]. PcDNA3.1-HA-Derlin was generously provided by Dr Yihong Ye [26]. Polyclonal anti-SVIP antibodies were generated by immunizing rabbit with purified glutathione S-transferase (GST)-SVIP protein. Anti-p97/VCP and anti-calnexin antibodies were purchased from Affinity BioReagents. Antigp and anti-Hrd antibodies have been previously described [25]. Polyclonal anti-Derlin and anti-VIMP antibodies were kindly provided by Dr Yihong Ye [26]. Anti-Npl antibodies were generated in rabbits using purified His6-Npl. Rabbit polyclonal anti-human ␣-1-antitrypsin was purchased from Biomeda. Human horseradish peroxidase-conjugated anti-␣1-antitrypsin was acquired from Bethyl Laboratories

Stable Cell Lines

Subcellular Fractionation

RESULTS

DISCUSSION