title: Insig-mediated, sterol-accelerated degradation of the membrane domain of hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase in insect cells.
abstract: Sterol-accelerated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase is one of several mechanisms through which cholesterol synthesis is controlled in mammalian cells. This degradation results from sterol-induced binding of the membrane domain of reductase to endoplasmic reticulum membrane proteins called Insig-1 and Insig-2, which are carriers of a ubiquitin ligase called gp78. The ensuing gp78-mediated ubiquitination of reductase is a prerequisite for its rapid, 26 S proteasome-mediated degradation from endoplasmic reticulum membranes, a reaction that slows a rate-limiting step in cholesterol synthesis. Here, we report that the membrane domain of hamster reductase is subject to sterol-accelerated degradation in Drosophila S2 cells, but only when mammalian Insig-1 or Insig-2 are co-expressed. This degradation mimics the reaction that occurs in mammalian cells with regard to its absolute requirement for the action of Insigs, sensitivity to proteasome inhibition, augmentation by nonsterol isoprenoids, and sterol specificity. RNA interference studies reveal that this degradation requires the Drosophila Hrd1 ubiquitin ligase and several other proteins, including a putative substrate selector, which associate with the enzyme in yeast and mammalian systems. These studies define Insigs as the minimal requirement for sterol-accelerated degradation of the membrane domain of reductase in Drosophila S2 cells.
authors.affiliation: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Nguyen
authors.forename: Soo Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Russell A
authors.initials: RA
authors.lastname: DeBose-Boyd
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004331:Drosophila melanogaster; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D013261:Sterols / Q000494:pharmacology*; D014162:Transfection; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D015853:Cysteine Proteinase Inhibitors; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D008565:Membrane Proteins; D061988:Proteasome Inhibitors; D013261:Sterols; D006903:Hydroxymethylglutaryl CoA Reductases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde