title: Dislocation of HMG-CoA reductase and Insig-1, two polytopic endoplasmic reticulum proteins, en route to proteasomal degradation.
abstract: The endoplasmic reticulum (ER) glycoprotein HMG-CoA reductase (HMGR) catalyzes the rate-limiting step in sterols biosynthesis. Mammalian HMGR is ubiquitinated and degraded by the proteasome when sterols accumulate in cells, representing the best example for metabolically controlled ER-associated degradation (ERAD). This regulated degradation involves the short-lived ER protein Insig-1. Here, we investigated the dislocation of these ERAD substrates to the cytosol en route to proteasomal degradation. We show that the tagged HMGR membrane region, HMG(350)-HA, the endogenous HMGR, and Insig-1-Myc, all polytopic membrane proteins, dislocate to the cytosol as intact full-length polypeptides. Dislocation of HMG(350)-HA and Insig-1-Myc requires metabolic energy and involves the AAA-ATPase p97/VCP. Sterols stimulate HMG(350)-HA and HMGR release to the cytosol concurrent with removal of their N-glycan by cytosolic peptide:N-glycanase. Sterols neither accelerate dislocation nor stimulate deglycosylation of ubiquitination-defective HMG(350)-HA((K89 + 248R)) mutant. Dislocation of HMG(350)-HA depends on Insig-1-Myc, whose dislocation and degradation are sterol independent. Coimmunoprecipitation experiments demonstrate sterol-stimulated association between HMG(350)-HA and Insig-1-Myc. Sterols do not enhance binding to Insig-1-Myc of HMG(350)-HA mutated in its sterol-sensing domain or of HMG(350)-HA((K89 + 248R)). Wild-type HMG(350)-HA and Insig-1-Myc coimmunoprecipitate from the soluble fraction only when both proteins were coexpressed in the same cell, indicating their encounter before or during dislocation, raising the possibility that they are dislocated as a tightly bound complex.
authors.affiliation: Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Gil S
authors.initials: GS
authors.lastname: Leichner
authors.forename: Rachel
authors.initials: R
authors.lastname: Avner
authors.forename: Dror
authors.initials: D
authors.lastname: Harats
authors.forename: Joseph
authors.initials: J
authors.lastname: Roitelman
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013261:Sterols / Q000494:pharmacology; D013816:Thermodynamics; D013997:Time Factors; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013261:Sterols; D006903:Hydroxymethylglutaryl CoA Reductases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C404942:p97 ATPase