Abstract
Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism*
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Amino Acid Sequence
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Amino Acid Substitution
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Animals
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Cell Line
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Cell Membrane / metabolism
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Cholesterol / metabolism
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Endoplasmic Reticulum / metabolism*
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Fatty Acids / chemistry
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Fatty Acids / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Molecular Sequence Data
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Proteasome Endopeptidase Complex / metabolism*
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Protein Subunits / genetics
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Protein Subunits / metabolism
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RNA Interference
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Receptors, Autocrine Motility Factor
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Receptors, Cytokine / genetics
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Receptors, Cytokine / metabolism
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Sequence Alignment
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination
Substances
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Fatty Acids
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INSIG1 protein, human
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INSIG2 protein, human
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Intracellular Signaling Peptides and Proteins
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Membrane Proteins
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Nuclear Proteins
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Protein Subunits
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Receptors, Cytokine
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Cholesterol
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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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p97 ATPase