Sequential Actions of the AAA-ATPase Valosin-containing Protein (VCP)/p97 and the Proteasome 19 S Regulatory Particle in Sterol-accelerated,Endoplasmic Reticulum (ER)-associated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase |
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Authors: | Lindsey L. Morris Isamu Z. Hartman Dong-Jae Jun Joachim Seemann Russell A. DeBose-Boyd |
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Affiliation: | From the Departments of ‡Molecular Genetics and ;§Cell Biology and ;the ¶Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046 |
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Abstract: | Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A 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 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (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 were 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 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates. |
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Keywords: | Cholesterol Metabolism Endoplasmic Reticulum (ER) Endoplasmic Reticulum-associated Protein Degradation (ERAD) Membrane Trafficking Proteasome ER-associated Degradation Cytosolic Dislocation Membrane Extraction |
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