Regulated endoplasmic reticulum-associated degradation of a polytopic protein: p97 recruits proteasomes to Insig-1 before extraction from membranes

J Biol Chem. 2009 Dec 11;284(50):34889-900. doi: 10.1074/jbc.M109.044875. Epub 2009 Oct 8.

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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Cell Line
  • Cell Membrane / metabolism
  • Cholesterol / metabolism
  • Endoplasmic Reticulum / metabolism*
  • Fatty Acids / chemistry
  • Fatty Acids / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Interference
  • Receptors, Autocrine Motility Factor
  • Receptors, Cytokine / genetics
  • Receptors, Cytokine / metabolism
  • Sequence Alignment
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • Fatty Acids
  • INSIG1 protein, human
  • INSIG2 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Nuclear Proteins
  • Protein Subunits
  • Receptors, Cytokine
  • Cholesterol
  • AMFR protein, human
  • Receptors, Autocrine Motility Factor
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex
  • Adenosine Triphosphatases
  • p97 ATPase