The SREBP pathway : Regulation of cholesterol metabolism by two step proteolysis of a membrane-bound transcription factor and molecular identification of sterol-regulated sitel-protease

Objective: Animal cells must regulate their cholesterol biosynthesis and uptake to supply sufficent amounts of cholesterol without risking overproduction. This coordination is achieved by a family of membrane-bound transcription factors called sterol regulatory element binding proteins (SREBPs). To enhance transcrtiption of genes encoding enzymes of cholesterol and fatty acid biosynthesis, the active NH2 terminal domain of SREBP is released from membranes of endoplasmic reticulum (ER) by two sequential cleavages. The first, regulated by sterols, is catalyzed Site-1 protease (S1P) that cleaves luminal loop of SREBPs. This reaction is mediated by a polytopic membrane protein called SREBP cleavage activating protein (SCAP) that complexes with SREBPs. SCAP appears to be a regulatory protein and serves as a sterol sensor. The second, not regulated by sterols, catalyzed by a hydrophobic zinc metallprotease, cleaves SREBPs within the first trans-membrane domain. Excess cholesterol block S1P activity thereby inhibits SREBP processing.<br>A cruial component of this regulatory pathway is the Site-1 protease (S1P) that initiates sterol-regulated release from the ER by making the first cut in the SREBPs. S1P is the target of the feedback regulation, but nothing is known about the structure and properties of S1P. To identify S1P, we carried out expression cloning.<br>Results: To clone S1P, we newly developed pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol deprived cells, cleavage by both proteases leads to PLAP secretion, but PLAP was not secreted in S1P deficient cells (SRD12B cells). We transfected pCMV-PLAP-BP2 plus pools of CHO cDNAs into SRD12B cells and monitored the PLAP secretion. We succeeded in identifying a cDNA that restores Site-1 cleavage. The cDNA encodes S1P; an intraluminal 1052-amino acid membrane bound subtilisin-like protease. We concluded that S1P is the sterol-regulated luminal protease that cleaves SREBPs and controls lipid metabolism in animal cells.