Direct Demonstration That Loop1 of Scap Binds to Loop7 A CRUCIAL EVENT IN CHOLESTEROL HOMEOSTASIS

Yinxin Zhang; Kwang Min Lee; Lisa N. Kinch; Lindsay Clark; Nick V. Grishin; Daniel M. Rosenbaum; Michael S. Brown; Joseph L. Goldstein; Arun Radhakrishnan

doi:10.1074/jbc.M116.729798

Direct Demonstration That Loop1 of Scap Binds to Loop7

A CRUCIAL EVENT IN CHOLESTEROL HOMEOSTASIS*

From the Departments of ^‡Molecular Genetics,
^¶Biophysics, and
^‖Biochemistry and
^§Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390

↵3 To whom correspondence may be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-2179; Fax: 214-648-8804; E-mail: mike.brown{at}utsouthwestern.edu.
↵4 To whom correspondence may be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-2141; Fax: 214-648-8804; E-mail: joe.goldstein{at}utsouthwestern.edu.
↵5 To whom correspondence may be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-0329; Fax: 214-648-8804; E-mail: arun.radhakrishnan{at}utsouthwestern.edu.

↵1 Present address: Laboratory of Molecular Genetics and Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065.
↵2 Present address: Korea Food Research Institute, Seongnam-si, Gyeonggi-do 13539, Republic of Korea.

Abstract

Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM HL20948 (to J. L. G. and M. S. B.), GM094575 (to N. V. G.), and R01GM113050 (to D. M. R.); Robert A. Welch Foundation Grants I-1793 (to A. R.), I-1505 (to N. V. G.), and I-1770 (to D. M. R.); and American Heart Association Grants 12SDG12040267 (to A. R.) and 16PRE27200004 (to L. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.