Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.
SREBPs exit the ER in a complex with SCAP. Together, they move to the Golgi where SREBP is cleaved, releasing a fragment that activates genes encoding lipid biosynthetic enzymes. Sterols block ER exit, preventing cleavage, decreasing transcription, and achieving feedback control of lipid synthesis. Here, we report an in vitro system to measure incorporation of SCAP into ER vesicles. When membranes were isolated from sterol-depleted cells, SCAP entered vesicles in a reaction requiring nucleoside triphosphates and cytosol. SCAP budding was diminished in membranes from sterol-treated cells. Kinetics of induction of budding in vitro matched kinetics of ER exit in living cells expressing GFP-SCAP. These data localize the sterol-regulated step to budding of SCAP from ER and provide a system for biochemical dissection.
Nohturfft
A
A
Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75390, USA.
Yabe
D
D
Goldstein
J L
JL
Brown
M S
MS
Espenshade
P J
PJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000494:pharmacology; D014157:Transcription Factors
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C102196:SREBP cleavage-activating protein; C497389:Srebf1 protein, rat; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D002784:Cholesterol