title: Membrane topology of human insig-1, a protein regulator of lipid synthesis.
abstract: Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from approximately 5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterol-sensing domains in SCAP and HMG CoA reductase.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
authors.forename: Jamison D
authors.initials: JD
authors.lastname: Feramisco
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004622:Embryo, Mammalian; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney; D008055:Lipids / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D013261:Sterols / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
chemical_list: C108626:INSIG1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C102196:SREBP cleavage-activating protein; D013261:Sterols; D025801:Ubiquitin; D006903:Hydroxymethylglutaryl CoA Reductases; D010450:Endopeptidases; D003545:Cysteine