Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis.
Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells.
Gong
Yi
Y
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
Lee
Joon No
JN
Brown
Michael S
MS
Goldstein
Joseph L
JL
Ye
Jin
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D017354:Point Mutation
C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; D001224:Aspartic Acid; D002784:Cholesterol