Autocatalytic Processing of Site-1 Protease Removes Propeptide and Permits Cleavage 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 lumen, thereby initiating a process by which the transcriptionally active NH2-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 assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH2-terminal signal sequence of S1P generates S1P-A (amino acids 23–1052), which is 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 Site-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 S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it 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 processing of a subtilisin-related protease that controls the level of cholesterol 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 characterize the activity of purified recombinant S1P. Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH2-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 assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH2-terminal signal sequence of S1P generates S1P-A (amino acids 23–1052), which is 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 Site-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 S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it 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 processing of a subtilisin-related protease that controls the level of cholesterol 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 characterize the activity of purified recombinant S1P. Cholesterol metabolism in animal cells is controlled by the sterol-regulated proteolysis of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; CMV, cytomegalovirus; endo H, endoglycosidase H; ER, endoplasmic reticulum; HEK-293 cells, human embryonic kidney 293 cells; PAGE, polyacrylamide gel electrophoresis; S1P, Site-1 protease; S2P, Site-2 protease; SCAP, SREBP cleavage-activating protein; TK, thymidine kinase; kb, kilobase(s) The sterol-regulated reaction is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves SREBPs in a hydrophilic loop that projects into the lumen of the endoplasmic reticulum (ER) and other organelles (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). An understanding of the mechanism of regulation of S1P is essential if we are to understand how animals control the cholesterol content of cells and blood. SREBPs are a family of three proteins, each of ∼1150 amino acids in length. After synthesis, each SREBP is inserted into the membranes of the ER and nuclear envelope in a hairpin orientation (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). The NH2-terminal segment of ∼480 amino acids is a transcription factor of the basic helix-loop-helix-leucine zipper family that projects into the cytoplasm. The middle segment of ∼80 amino acids consists of two membrane-spanning helices separated by a luminal hydrophilic loop of ∼30 amino acids. The COOH-terminal segment of ∼590 amino acids extends into the cytoplasm, where it forms a complex with the COOH-terminal segment of a membrane-bound regulatory protein designated SREBP-cleavage activating protein (SCAP). The SREBP/SCAP complex is the true substrate for S1P; disruption of this complex in intact cells abrogates the proteolytic reaction (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar,3Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 5785-5793Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). S1P initiates the processing of SREBPs by cleaving at a site in the middle of the luminal loop. This reaction has been studied most extensively for human SREBP-2. S1P cleaves this protein between the leucine and serine of the sequence RSVLS (4Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The specificity of recognition has been studied by transfecting cDNAs encoding mutant forms of SREBP-2 into cultured cells. Cleavage of SREBP-2 absolutely requires arginine or lysine at the P4 position. Although the full range of residues at the P1 position not cleavage the leucine at P1 by or by the The serine at the and the serine and at the and be by with Cleavage by S1P the SREBP into two each of which has a membrane-spanning This a designated Site-2 protease to the NH2-terminal at a position membrane-spanning sequence R.B. D. J. Sakai J. Brown M.S. Goldstein J.L. Mol. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google E.A. Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). This cleavage the NH2-terminal segment of SREBP, it to the where it to and transcription of encoding the and of cholesterol and acid (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). in cells, the cleavage of SREBPs by S1P is SREBPs to and transcription of the This regulation is by the of (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Nohturfft A. Goldstein J.L. Brown M.S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism by which the Site-1 cleavage reaction and the mechanism by which this is S1P by in cells, a mutant of hamster cells that is to cholesterol or to The in cells to a in the Site-1 cleavage reaction R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and this the cells transfected with of cDNAs from hamster cells (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The and to S1P. analysis showed that the cells in the S1P that the of S1P data that the sequence of human S1P been a of a in A. 2: PubMed Scopus Google Scholar). The hamster and human proteins are (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). S1P is a of amino acids. The NH2-terminal amino acids are and a signal This is by a of amino acids that S1P a subtilisin-related protease (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). with other family it to the and serine (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google 1997; PubMed Scopus Google M.S. Goldstein J.L. A. Scholar). of of amino acids S1P to the in cells, that residues are for activity (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). the is a of amino acids that has to other This is by a sequence of amino acids that appears to be a membrane-spanning and a COOH-terminal of amino acids that is to into the This sequence is because it is in and basic that S1P is membrane-bound (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The of subtilisin-related that are in 1997; PubMed Scopus Google Scholar). The of this family of and the which in the or to process to or to the J. 1997; PubMed Scopus Google Scholar). of basic a basic at the P4 position. The recognition sequence is where for amino acid J. 1997; PubMed Scopus Google Scholar). S1P differs from the of the family because it cleaves a amino In this it of the by from M. PubMed Scopus Google Scholar). J. A. J. M. C. M. M. A. PubMed Scopus Google the of a human subtilisin-related called which is to S1P. These in a for proteins that In experiments, that cleaved the of a secreted the of the sequence The of this cleavage is not because this cleavage site not to the site at which factor is J. A. M. P. PubMed Scopus Google L. M. J. L. PubMed Scopus Google Scholar). of the family are that are by a cleavage reaction that an NH2-terminal propeptide 1997; PubMed Scopus Google J. 1997; PubMed Scopus Google Scholar). J. A. J. M. C. M. M. A. PubMed Scopus Google that a processing reaction an in the site of cleavage not In the current we that S1P is an membrane-bound and that it that active The most cleavage the sequence and appears to be In an accompanying we a secreted of S1P and D. Espenshade P.J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). from from or from from from from from and from from (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google D. Espenshade P.J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Brown M.S. Goldstein J.L. Cell. Full Text PDF PubMed Scopus Google A. Brown M.S. Goldstein J.L. A. 1998; PubMed Scopus Google Scholar). and J.L. Brown M.S. PubMed Scopus Google Scholar). The the of is a that hamster with a that the protein to Nohturfft A. Goldstein J.L. Brown M.S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google which hamster S1P; which hamster S1P three of the inserted between amino acids and and which is to that serine has been to is to that amino acids of S1P been from to is to that amino acids and been to and and we used the to a of into to the used to and for the Site-B and and for the the to an of to yield the both the Site-B and and by hamster S1P (amino acids three of the at the by of this is in the accompanying in which it is to D. Espenshade P.J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is to that serine has been to a of to a of to a of to a of to The the thymidine from of the and by a by of to a by from is to that amino acids been from to is to that amino acids and been to and both Site-B and from or to a from to and human SREBP-2 with two of the at the and has been Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and mutant of in which the recognition sequence for S1P in human SREBP-2 has been to and These two by (4Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). a a of human SREBP-2 to a of by this we amino acids of SREBP-2 to or RRLL with the or fragments of SREBP-2 the into to and In by the the which of transfected cDNAs C. Mol. Cell. Biol. PubMed Scopus Google Scholar). amino acids of hamster S1P (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google by with a of two to amino acids and each with an NH2-terminal that to a D. Scholar). of human embryonic kidney 293 cells for and cultured in at in and with the cells transfected an with the Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the cells with of with and cultured for to The from two and at at for An of the with of and at for The proteins at at for and in of at The from J. E.A. Rawson R.B. Brown M.S. Goldstein J.L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of cells R.B. Cheng D. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and cultured in at in of and and with and the cells transfected with the to the (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). the cells with of with The cells for to each at a of and the cells cells from used to nuclear and J. E.A. Rawson R.B. Brown M.S. Goldstein J.L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in which cells not at a of with at and and at for to proteins to with in the and by (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). or with a HEK-293 cells cultured for in of with which membranes and with in the or of for at A. Brown M.S. Goldstein J.L. A. 1998; PubMed Scopus Google Scholar). to by from transfected cells which of protein with or endoglycosidase (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). cells and cells transfected with D. Espenshade P.J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in and at in with The other the to with the cells and with The of in of at with and The for at by at for at The with of to with for at The by for at The and the with of A. The further by at in of which the in of and into of the with of A. in of PubMed Scopus Google for and at of the to and with or to a and with The that in from the cells each from the and to NH2-terminal sequence analysis by with from and by the the of the of S1P and the position of three internal cleavage that to forms of S1P, designated and C. we cDNAs that epitope-tagged of S1P control of the These of S1P with of a at the or the The NH2-terminal inserted the site of cleavage by signal that it with the protein this The COOH-terminal by which of the protein a a of the SREBP/SCAP complex and the position of S1P with active site to the cleavage site in that the bulk of S1P is in the ER lumen, we protease protection assays and HEK-293 cells transfected with cDNAs encoding or and with of in the or of the the at the the not by of intact it the membranes with that this of the protein is the other the at the it by both in the and of the and that the we used an to that an from in the of in and protein by the to in the of because of of this a that has been Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). These data the orientation of S1P that is in the processing of S1P, we used cells that lack S1P. transfected the cells with and studied the of the by with an the of S1P The three that we and protein from we transfected the cells with in which the serine at the active site to In this S1P-A that processing to the and forms requires a active site and is the the of S1P, the data that S1P-B and S1P-C been by proteolytic of NH2-terminal an to the of the NH2-terminal propeptide the proteolytic processing of S1P. this we used HEK-293 cells, which yield of protein from transfected cDNAs cells. transfected HEK-293 cells with and of the and a to and with an the NH2-terminal The membranes a to S1P-A The a at This not we transfected the epitope-tagged mutant of S1P that it by that this secreted protein the NH2-terminal propeptide that is cleaved to S1P-B S1P of that are by in A. cells transfected with the of and S1P-C with that the membranes with endo H, the of S1P-A and S1P-B the of S1P-C not a endo S1P-B and S1P-C the gel These that the of S1P-A and S1P-B in the endo of ER proteins, S1P-C processing by the S1P at which processing we transfected cells with which S1P with a at the of transfected cells, designated by in the of and the cells cultured in bulk in and proteins with to and with a we from cells that by of cholesterol and acids to the medium. The from the cells that not in the control cells. of from a and at the NH2-terminal by of the in to and The that S1P-A is the of signal cleavage at the S1P-B from cleavage the sequence S1P-C from cleavage the sequence of are in the human and hamster proteins the two the gel that and two protein that may with S1P, with ER proteins A. Biol. PubMed Scopus Google Scholar). the and forms of S1P are we cDNAs encoding mutant forms of S1P with at the and cleavage In the Site-B the sequence at the cleavage site to In the we the P4 to of the cleavage site from to and we a sequence that an cleavage site at residues to The cDNAs into cells by and the forms of S1P to and by with the the COOH-terminal of S1P. in the encoding S1P to and S1P-C the mutant showed the S1P-A at Site-B and it S1P-C The mutant to it of S1P-B the cleavage of S1P from the NH2-terminal we transfected the and mutant into HEK-293 cells and studied the of the propeptide by and of the with the the NH2-terminal In the the of S1P, in cells transfected with the S1P and of the The membranes of a to the propeptide the mutant The the propeptide the cells S1P or the mutant not the cells the mutant or the Site-B mutant the data of to that the of S1P is cleaved at Site-B and at Site-B cleavage is in the Site-B cleavage at is the other in the not with Site-B Site-B cleavage a propeptide is This protein with a recombinant protein that to amino acids of S1P not used the Site-B and to or is active in cleaving SREBP-2 this we transfected cells with a encoding a of human SREBP-2 with an at the cDNAs encoding S1P or of the and nuclear to and the of SREBP-2 by with In the of S1P, the membranes the of nuclear The nuclear we of the mutant of S1P a transfected The Site-B mutant to the cleaved nuclear of this activity of of the Site-B mutant with S1P and the other the mutant active the S1P with that of each of the S1P to of S1P-A S1P to the and which not with the mutant or the Site-B The mutant to the and forms of S1P. of the data in and that S1P cleavage of SREBP-2 at a at which the of S1P and this to the of the and the that S1P that a of active is to of the SREBP-2. the cleavage activity of we the SREBP-2 cleavage assay a mutant S1P that both the Site-B and designated cells transfected with the mutant S1P-A by with at both Site-B and processing of In to the for the Site-B and and of cells with SREBP-2 and the mutant of S1P in cleaved nuclear of SREBP-2 These data that S1P-A is an of S1P that requires the of the NH2-terminal propeptide in to the two active S1P-B and that SREBP-2. The data in and that S1P-B and S1P-C are by this is S1P be to to Site-B and and in to the RSVL sequence that is cleaved in SREBP-2. this we cDNAs encoding mutant forms of SREBP-2 with the Site-B and recognition for the RSVL sequence These cDNAs transfected into cells with or a encoding S1P. and nuclear to and with The data that S1P is to SREBP-2 the Site-B sequence and the sequence the sequence cleavage of the sequence we the arginine at the P4 position with Cleavage of the Site-B and by the arginine to at P4 not The in to each S1P cleaves in an intramolecular reaction to the and forms or active S1P cleaves in an transfected cells with a encoding S1P or the In we the or of to and with the the of S1P. this not with S1P that has a at the to the S1P. the cells S1P, and S1P-A the cells the mutant S1P S1P-C S1P-B not The S1P by not yield a These that an active S1P S1P at not at cleavage at Site-B are controls that that the mutant not to the mutant S1P to the with the which that the S1P to the and forms and not it the and S1P be to the or in to be it is that S1P activity by the processing this we transfected cells with a encoding SREBP-2 The cells or with with and nuclear with The processing of S1P to the and forms in the and of and the other nuclear SREBP-2 in the of These data that not the of S1P to active or the of the active to The current data provide that S1P, in the is an that cleavage in to an active The of S1P by a of protease protection assays and protein These that S1P a acid NH2-terminal signal sequence that the protein into the lumen of the ER and is by signal to the designated S1P-A to ER membranes by of the sequence the This sequence is by a sequence that projects into the The cleavage of S1P at Site-B, which is the in the sequence This cleavage a propeptide from the of This cleavage not the serine of S1P is with that the cleavage is The mutant is not cleaved to the cells other of active S1P, that S1P-B be by an intramolecular cleavage reaction the propeptide is not it is secreted from the that the secreted propeptide is the of cleavage at Site-B from three of S1P at Site-B the of this the of this is to that of a recombinant protein to amino acids of S1P not and the secreted propeptide is not by that it not not the propeptide by cleavage at it at After the propeptide is released by cleavage at Site-B, S1P-B further cleavage at which is the in the sequence This cleavage requires an active S1P because is by the in to Site-B the cleavage reaction be catalyzed by a S1P in an reaction The processing reaction in this paper to and from the processing of and the which been studied extensively J. 1997; PubMed Scopus Google Scholar). S1P, are inserted into the ER lumen by of a signal and are anchored to the by a sequence the In the ER, process to of amino acids that are in not in to the 115-amino acid propeptide released from S1P J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the propeptide with the activity the the where a cleavage the it to from the which is thereby J. 1997; PubMed Scopus Google Scholar). This cleavage reaction requires and that in the J. 1997; PubMed Scopus Google Scholar). In to the with data that the S1P propeptide is not further it is secreted by After the propeptide is S1P-B endo that Site-B cleavage in the be to the propeptide is released from S1P cleavage at Site-B or it thereby the The to the propeptide be data that the of S1P-A to S1P-B and S1P-C is not by it that the of the propeptide from S1P-B is by and that this regulation a in the activity of S1P. The that S1P cleaves at an sequence and that it is of cleaving a mutant SREBP-2 with at the cleavage site the to (4Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This is in to the sequence specificity of and the in which an arginine at P1 is essential J. 1997; PubMed Scopus Google Scholar). data that S1P cleaves SREBP-2 in a (4Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In experiments, for into and Site-1 cleavage to in an endo (4Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This that the active of S1P be endo The two forms of S1P that are endo are S1P-A and S1P-B S1P-A is by the of cleavage activity both Site-B and are it that most of the Site-1 cleavage of SREBP-2 is by that the activity of the Site-B mutant is to the S1P-C that forms In an accompanying paper D. Espenshade P.J. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we a secreted of S1P-C and that it is active in S1P-C is in the ER, it be transported to the because are endo S1P-C may be to a of SREBP-2 it the S1P differs from and the in the in for an (2Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). be to S1P or is to SREBPs to the site that active S1P. that between the ER and in cells and that to be in the ER A. Brown M.S. Goldstein J.L. A. 1998; PubMed Scopus Google Scholar). This a in which SREBPs to a cleavage site that is in between the ER and the Golgi, where active S1P and for for in for protein and for with and for and Sakai and for and this