Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.
We report the isolation and characterization of a new line of mutant Chinese hamster ovary cells, designated SRD-5, that are resistant to 25HC, a potent suppressor of cleavage of sterol regulatory element-binding proteins (SREBPs) in mammalian cells. In SRD-5 cells, SREBPs are cleaved constitutively, generating transcriptionally active nuclear SREBP even in the presence of sterols. Sequence analysis of SREBP cleavage-activating protein (SCAP) transcripts from SRD-5 cells revealed the presence of a mutation in one SCAP allele that results in substitution of a conserved Leu by Phe at amino acid 315 within the sterol-sensing domain. Sterols fail to inhibit the packaging of SREBPSCAP(L315F) complexes into budding vesicles in vitro. Sterols also fail to induce binding of SCAP(L315F) to insig-1 or insig-2, two proteins that function in the sterol-mediated retention of SREBPSCAP complexes in the endoplasmic reticulum. Similar findings were observed for SCAP(D443N) and SCAP(Y298C), both of which cause a sterol-resistant phenotype. Thus, three different point mutations, each within the sterol-sensing domain of SCAP, prevent sterol-induced binding of SCAP to insig proteins and abolish feedback regulation of SREBP processing by sterols.
Yabe
Daisuke
D
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Xia
Zong-Ping
ZP
Adams
Christopher M
CM
Rawson
Robert B
RB
D000595:Amino Acid Sequence; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C102196:SREBP cleavage-activating protein; C497388:Srebf1 protein, mouse; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors