Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism.
Sterol accumulation in membranes blocks the exit of SCAP from the ER, preventing SREBP cleavage and reducing cholesterol synthesis. Sterols act through SCAP's sterol-sensing domain by an obscure mechanism. Here, we show that addition of cholesterol to ER membranes in vitro causes a conformational change in SCAP, detected by the unmasking of closely spaced trypsin cleavage sites. Two mutant forms of SCAP (Y298C and D443N) that are refractory to sterol regulation in vivo are also refractory to sterol-induced conformational change in vitro. 25-hydroxycholesterol, a potent regulator of SCAP in vivo, fails to change SCAP's conformation in vitro, suggesting that oxysterols act in intact cells by translocating cholesterol from plasma membrane to ER. These studies demonstrate an in vitro effect of cholesterol on the sterol regulatory machinery.
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Andrew J
AJ
Brown
Liping
L
Sun
Jamison D
JD
Feramisco
Michael S
MS
Brown
Joseph L
JL
Goldstein
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology*; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C497390:SREBF2 protein, human; C102196:SREBP cleavage-activating protein; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; D019308:Palmitic Acid; D002784:Cholesterol; D014357:Trypsin