title: Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.
abstract: Accumulation of sterols in membranes of the endoplasmic reticulum (ER) leads to the accelerated ubiquitination and proteasomal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids. This degradation results from sterol-induced binding of reductase to the Insig-1 or Insig-2 proteins of ER membranes. We previously reported that in immortalized human fibroblasts (SV-589 cells) Insig-1, but not Insig-2, recruits gp78, a membrane-bound RING-finger ubiquitin ligase. We now report that both Insig-1 and Insig-2 bind another membrane-bound RING-finger ubiquitin ligase called Trc8. Knockdown of either gp78 or Trc8 in SV-589 cells through RNA interference (RNAi) inhibited sterol-induced ubiquitination of reductase and inhibited sterol-induced degradation by 50-60%. The combined knockdown of gp78 and Trc8 produced a more complete inhibition of degradation (> 90%). Knockdown of gp78 led to a three to fourfold increase in levels of Trc8 and Insig-1 proteins, which opposed the inhibitory action of gp78. In contrast, knockdown of Trc8 had no effect on gp78 or Insig-1. The current results suggest that sterol-induced ubiquitination and proteasomal degradation of reductase is dictated by the complex interplay of at least four proteins: Insig-1, Insig-2, gp78, and Trc8. Variations in the concentrations of any one of these proteins may account for differences in cell- and/or tissue-specific regulation of reductase degradation.
authors.affiliation: Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
authors.forename: Youngah
authors.initials: Y
authors.lastname: Jo
authors.forename: Peter C W
authors.initials: PC
authors.lastname: Lee
authors.forename: Peter V
authors.initials: PV
authors.lastname: Sguigna
authors.forename: Russell A
authors.initials: RA
authors.lastname: DeBose-Boyd
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D034622:RNA Interference; D060285:Receptors, Autocrine Motility Factor / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013261:Sterols / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
chemical_list: C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C114025:RNF139 protein, human; D011956:Receptors, Cell Surface; D013261:Sterols; D006903:Hydroxymethylglutaryl CoA Reductases; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases