Autocatalytic processing of site-1 protease removes propeptide and permitscleavage of sterol regulatory element-binding proteins

Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum l umen, thereby initiating a process by which the transcriptionally active NH 2-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P, Protease protection as says showed that the bulk of S1P is in the endoplasmic reticulum lumen, anc hored by a COOH-terminal membrane-spanning segment. Cleavage of the NH2-ter minal signal sequence of S1P generates S1P-A (amino acids 23-1052), which i s inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Si te-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2, S1P-B is further cleaved at an internal RRLL sequence to yield S1P -C (amino acids 187-1052), Mutational analysis suggests that S1P-B and SIP- C are both active in cleaving SREBP-2 in a fashion that requires SREBP clea vage-activating protein, The activity of S1P-C may be short-lived because i t appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur, These data provide the initial description of the p rocessing of a subtilisin-related protease that controls the level of chole sterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. ( 1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to c haracterize the activity of purified recombinant S1P.