Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein.
We describe a line of mutant Chinese hamster ovary cells, designated SRD-13A, that cannot cleave sterol regulatory element-binding proteins (SREBPs) at site 1, due to mutations in the gene encoding SREBP cleavage-activating protein (SCAP). The SRD-13A cells were obtained by two rounds of gamma-irradiation followed first by selection for a deficiency of low density lipoprotein receptors and second for cholesterol auxotrophy. In the SRD-13A cells, the only detectable SCAP allele encodes a truncated nonfunctional protein. In the absence of SCAP, the site 1 protease fails to cleave SREBPs, and their transcriptionally active NH(2)-terminal fragments cannot enter the nucleus. As a result, the cells manifest a marked reduction in the synthesis of cholesterol and its uptake from low density lipoproteins. The SRD-13A cells grow only when cholesterol is added to the culture medium. SREBP cleavage is restored and the cholesterol requirement is abolished when SRD-13A cells are transfected with expression vectors encoding SCAP. These results provide formal proof that SCAP is essential for the cleavage of SREBPs at site 1.
Rawson
R B
RB
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
DeBose-Boyd
R
R
Goldstein
J L
JL
Brown
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C102196:SREBP cleavage-activating protein; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D004247:DNA; D002784:Cholesterol