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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Adenosine Triphosphatases / antagonists & inhibitors
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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CD3 Complex / genetics
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CD3 Complex / metabolism
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Calnexin / genetics
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Calnexin / metabolism
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Carrier Proteins / antagonists & inhibitors
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Line
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Endoplasmic Reticulum / enzymology*
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Endoplasmic Reticulum / genetics
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Gene Silencing
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Humans
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Membrane Proteins / antagonists & inhibitors
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Phosphate-Binding Proteins
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Proteasome Endopeptidase Complex / genetics
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Proteasome Endopeptidase Complex / metabolism
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Proteasome Inhibitors*
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Protein Folding*
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Protein Processing, Post-Translational / physiology*
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Receptors, Autocrine Motility Factor
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Receptors, Cytokine / antagonists & inhibitors
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Receptors, Cytokine / genetics
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Receptors, Cytokine / metabolism
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Ubiquitin-Protein Ligases / antagonists & inhibitors
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination / physiology*
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Valosin Containing Protein
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alpha 1-Antitrypsin / genetics
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alpha 1-Antitrypsin / metabolism
Substances
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CD3 Complex
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CD3delta antigen
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Carrier Proteins
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Cell Cycle Proteins
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DERL1 protein, human
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Membrane Proteins
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Nuclear Proteins
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Phosphate-Binding Proteins
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Proteasome Inhibitors
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Receptors, Cytokine
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SVIP protein, human
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alpha 1-Antitrypsin
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Calnexin
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AMFR protein, human
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Receptors, Autocrine Motility Factor
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Ubiquitin-Protein Ligases
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Proteasome Endopeptidase Complex
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Adenosine Triphosphatases
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VCP protein, human
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Valosin Containing Protein