title: Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for sterol-regulated enzyme degradation.
abstract: The stability of the endoplasmic reticulum (ER) glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in cholesterol biosynthesis, is negatively regulated by sterols. HMGR is anchored in the ER via its N-terminal region, which spans the membrane eight times and contains a sterol-sensing domain. We have previously established that degradation of mammalian HMGR is mediated by the ubiquitin-proteasome system (Ravid, T., Doolman, R., Avner, R., Harats, D., and Roitelman, J. (2000) J. Biol. Chem. 275, 35840-35847). Here we expressed in HEK-293 cells an HA-tagged-truncated version of HMGR that encompasses all eight transmembrane spans (350 N-terminal residues). Similar to endogenous HMGR, degradation of this HMG(350)-3HA protein was accelerated by sterols, validating it as a model to study HMGR turnover. The degradation of HMG(240)-3HA, which lacks the last two transmembrane spans yet retains an intact sterol-sensing domain, was no longer accelerated by sterols. Using HMG(350)-3HA, we demonstrate that transmembrane region of HMGR is ubiquitinated in a sterol-regulated fashion. Through site-directed Lys --> Arg mutagenesis, we pinpoint Lys(248) and Lys(89) as the internal lysines for ubiquitin attachment, with Lys(248) serving as the major acceptor site for polyubiquitination. Moreover, the data indicate that the N terminus is also ubiquitinated. The degradation rates of the Lys --> Arg mutants correlates with their level of ubiquitination. Notably, lysine-less HMG(350)-3HA is degraded faster than wild-type protein, suggesting that lysines other than Lys(89) and Lys(248) attenuate ubiquitination at the latter residues. The ATP-dependent ubiquitination of HMGR in isolated microsomes requires E1 as the sole cytosolic protein, indicating that ER-bound E2 and E3 enzymes catalyze this modification. Polyubiquitination of HMGR is correlated with its extraction from the ER membrane, a process likely to be assisted by cytosolic p97/VCP/Cdc48p-Ufd1-Npl4 complex, as only ubiquitinated HMGR pulls down p97.
authors.affiliation: Institute of Lipid and Atherosclerosis Research, Sheba Medical Center, Tel Hashomer 52621, Israel.
authors.forename: Ram
authors.initials: R
authors.lastname: Doolman
authors.forename: Gil S
authors.initials: GS
authors.lastname: Leichner
authors.forename: Rachel
authors.initials: R
authors.lastname: Avner
authors.forename: Joseph
authors.initials: J
authors.lastname: Roitelman
mesh_terms: D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D013261:Sterols; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D001120:Arginine; D006903:Hydroxymethylglutaryl CoA Reductases; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; D008239:Lysine