Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.
SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.
Nohturfft
A
A
Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
Brown
M S
MS
Goldstein
J L
JL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004351:Drug Resistance; D005038:Ethylnitrosourea; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006888:Hydroxycholesterols; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C497387:SREBF1 protein, human; C102196:SREBP cleavage-activating protein; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; C007997:25-hydroxycholesterol; D005038:Ethylnitrosourea