title: Point mutation in luminal loop 7 of Scap protein blocks interaction with loop 1 and abolishes movement to Golgi.
abstract: Scap is a polytopic protein of the endoplasmic reticulum (ER) that controls cholesterol homeostasis by transporting sterol regulatory element-binding proteins (SREBPs) from the ER to the Golgi complex. Scap has eight transmembrane helices (TM) joined by four small hydrophilic loops and three large loops. Two of the large loops (Loops 1 and 7) are in the ER lumen, and the other large loop (Loop 6) faces the cytosol where it binds COPII proteins that initiate transport to Golgi. Cholesterol binding to Loop 1 alters the configuration of Loop 6, precluding COPII binding and preventing the exit of Scap from the ER. Here, we create a point mutation (Y640S) in luminal Loop 7 that prevents Scap movement to Golgi. Trypsin cleavage assays show that Loop 6 of Scap(Y640S) is always in the configuration that precludes COPII binding, even in the absence of cholesterol. When expressed separately by co-transfection, the NH2-terminal portion of Scap (containing TM helices 1-6, including Loop 1) binds to the COOH-terminal portion (containing TM helices 7-8 and Loop 7) as determined by co-immunoprecipitation. This binding does not occur when Loop 7 contains the Y640S mutation. Co-immunoprecipitation is also abolished by a point mutation in Loop 1 (Y234A) that also prevents Scap movement. These data suggest that Scap Loop 1 must interact with Loop 7 to maintain Loop 6 in the configuration that permits COPII binding. These results help explain the operation of Scap as a sterol sensor.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046.
authors.forename: Yinxin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046.
authors.forename: Massoud
authors.initials: M
authors.lastname: Motamed
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046.
authors.forename: Joachim
authors.initials: J
authors.lastname: Seemann
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046. Electronic address: mike.brown@utsouthwestern.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046. Electronic address: joe.goldstein@utsouthwestern.edu.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003470:Culture Media; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D051779:Sterol Regulatory Element Binding Proteins / Q000378:metabolism; D013261:Sterols / Q000378:metabolism
chemical_list: D003470:Culture Media; D047908:Intracellular Signaling Peptides and Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; D051779:Sterol Regulatory Element Binding Proteins; D013261:Sterols; D002784:Cholesterol
keywords: Cholesterol Regulation; Cholesterol-binding Protein; Membrane Function; Membrane Lipids; Membrane Proteins; Scap; Sterol Regulatory Element-binding Protein (SREBP)
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