title: Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis.
abstract: Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.
authors.affiliation: From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
authors.forename: Yansong
authors.initials: Y
authors.lastname: Gao
authors.affiliation: From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
authors.forename: Yulian
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 joe.goldstein@utsouthwestern.edu.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
authors.affiliation: From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 mike.brown@utsouthwestern.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
authors.affiliation: From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 arun.radhakrishnan@utsouthwestern.edu.
authors.forename: Arun
authors.initials: A
authors.lastname: Radhakrishnan
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; D002784:Cholesterol
keywords: cholesterol regulation; cholesterol-binding protein; conformational change; membrane lipid; membrane protein