Point mutation in luminal Loop 7 of scap protein blocks interaction with Loop 1 and abolishes movement to Golgi

Yinxin Zhang, Massoud Motamed, Joachim Seemann, Michael S. Brown, Joseph L. Goldstein

Research output: Contribution to journalArticle

16 Scopus citations

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 NH 2-terminal portion of Scap (containingTMhelices 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 Loop1(Y234A) that also prevents Scapmovement.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.

Original languageEnglish (US)
Pages (from-to)14059-14067
Number of pages9
JournalJournal of Biological Chemistry
Volume288
Issue number20
DOIs
StatePublished - May 17 2013

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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