Sequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductase

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dc.contributor.advisorGoodman, Joel M.en
dc.contributor.committeeMemberDeBose-Boyd, Russell A.en
dc.contributor.committeeMemberLehrman, Mark A.en
dc.contributor.committeeMemberDe Martino, Georgeen
dc.creatorMorris, Lindsey LaChelleen
dc.date.accessioned2016-09-01T19:16:50Z
dc.date.available2016-09-01T19:16:50Z
dc.date.created2014-08
dc.date.issued2014-05-28
dc.date.submittedAugust 2014
dc.date.updated2016-09-01T19:07:40Z
dc.description.abstractAccelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme HMG CoA reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26S proteasomes through poorly defined reactions mediated by the AAA-ATPase VCP/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion, and thus membrane extraction of reductase, was tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.en
dc.format.mimetypeapplication/pdfen
dc.identifier.oclc957676349
dc.identifier.urihttps://hdl.handle.net/2152.5/3571
dc.language.isoenen
dc.subjectAdenosine Triphosphatasesen
dc.subjectEndoplasmic Reticulumen
dc.subjectHydroxymethylglutaryl CoA Reductasesen
dc.subjectMetalloendopeptidasesen
dc.subjectProteolysisen
dc.subjectSterolsen
dc.titleSequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductaseen
dc.typeThesisen
dc.type.materialtexten
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.disciplineCell Regulationen
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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