Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies.
Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP.SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP.SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction.
Sakai
J
J
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Nohturfft
A
A
Goldstein
J L
JL
Brown
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006031:Glycosylation; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011388:Prolactin / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C102196:SREBP cleavage-activating protein; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; D011388:Prolactin