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Author’s ChoiceLipid-regulated degradation of HMG-CoA reductase and Insig-1 by way of distinct mechanisms in insect cellsRebecca A. Faulkner, Andrew D. Nguyen, Youngah Jo, and Russell A. DeBose-BoydHoward Hughes Health-related Institute, Department of Molecular Genetics, University of Texas Southwestern Health-related Center, Dallas, TX 75390-Abstract In mammalian cells, levels on the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the very connected Insig-2 protein.Osilodrostat Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation.Poziotinib In contrast to reductase, Insig-1 is subjected to ERAD in lipiddeprived cells.PMID:23008002 Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by stopping the protein’s cytosolic dislocation. In previous research, we discovered that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and expected the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that happen to be mediated by different ubiquitin ligase complexes. With each other, these results establish Drosophila S2 cells as a model method to elucidate mechanisms via which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.– Faulkner, R. A., A. D. Nguyen, Y. Jo, and R. A. DeBose-Boyd. Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells. J. Lipid Res. 2013. 54: 1011022.Supplementary key words endoplasmic reticulum-associated degradation lipid metabolism ubiquitin ligase cytosolic dislocation 3-hydroxy-3-methylglutaryl-CoAIn mammalian cells, the endoplasmic reticulum-associated degradation (ERAD) pathway controls levels of two integral membrane proteins that play essential roles within the upkeep of lipid homeostasis, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and Insig-1. HMG-CoA reductase catalyzes the reduction of HMG-CoA to mevalonate, a ratelimiting reaction inside the synthesis of cholesterol and crucial nonsterol isoprenoids (1). Sterol accumulation triggers binding of reductase to either Insig-1 or its extremely connected isoform Insig-2 in endoplamic reticulum (ER) membranes (two). Insig binding is mediated completely by the membrane domain of reductase, which consists of eight membranespanning helice.