Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2

A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with 35Smethionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage. A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with 35Smethionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage. Proteolytic processing of sterol regulatory element-binding proteins (SREBPs) 1The abbreviations used are: SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticulum; LDL, low density lipoprotein; SCAP, SREBP cleavage-activating protein; PAGE, polyacrylamide gel electrophoresis; VAI, virus-associated I. controls the metabolism of cholesterol and fatty acids in animal cells (1Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). SREBPs are transcription factors that are bound to membranes of the ER and nuclear envelope. Each SREBP is composed of three segments: 1) an NH2-terminal segment of ∼485 amino acids that is a transcription factor of the basic helix-loop-helix-leucine zipper family, 2) a membrane attachment segment of ∼75 amino acids composed of two membrane-spanning sequences separated by a short hydrophilic loop of 31 amino acids, and 3) a COOH-terminal segment of ∼585 amino acids that plays a regulatory role. The proteins are oriented so that the NH2- and COOH-terminal segments project into the cytoplasm, and only the short hydrophilic loop projects into the lumen of the ER or nuclear envelope (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Before it can activate transcription, the NH2-terminal segment is released from the membrane in a complex two-step proteolytic sequence (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). First, a protease cleaves the protein at Site-1, which is near an arginine in the lumenal loop, thereby breaking the attachment between the two transmembrane sequences. This allows a second protease to cleave the protein at Site-2, which is near the middle of the first transmembrane sequence (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). The NH2-terminal fragment leaves the membrane with a portion of the first transmembrane sequence still attached. It enters the nucleus, where it activates transcription of genes encoding the LDL receptor (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar,6Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (501) Google Scholar), several enzymes of cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A synthase (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 6Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (501) Google Scholar), 3-hydroxy-3-methylglutaryl coenzyme A reductase (7Vallett S.M. Sanchez H.B. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1996; 271: 12247-12253Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), farnesyl diphosphate synthase (8Ericsson J. Jackson S.M. Edwards P.A. J. Biol. Chem. 1996; 271: 24359-24364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and squalene synthase (9Guan G. Jiang G. Koch R.L. Shechter I. J. Biol. Chem. 1995; 270: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)), and enzymes of fatty acid biosynthesis (10Kim J.B. Spiegelman B.M. Genes 10: 1096-1107Crossref PubMed Scopus (846) Google Scholar, 11Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and desaturation (12Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar). The net result is to increase the cell's supply of cholesterol and fatty acids. The Site-1 protease is the target of feedback regulation by cholesterol and other sterols (3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). When these sterols accumulate within cells, the rate of proteolysis at Site-1 declines markedly. Cleavage at Site-2 also declines because this cleavage requires prior cleavage at Site-1 (3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). As a result, the amounts of nuclear SREBPs decline, and transcription of the target genes falls. The net effect is to prevent overaccumulation of cholesterol and fatty acids when intracellular sterol levels are already high. Three isoforms of SREBP are known (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 13Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar, 14Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus Google Scholar). and are from a through use of that first (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 13Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar, 14Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus Google Scholar). is active in transcription of known target genes H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: PubMed Scopus Google Scholar). The is the of a X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (501) Google X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (247) Google Scholar), and it is also active H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: PubMed Scopus Google Scholar). of the regulatory of the Site-1 of and of regulation is A first be the of the site at which the Site-1 protease we that this cleavage is when arginine in the lumenal loop of SREBP-2 the arginine of is to by in mutagenesis (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The of the cleavage as by is with cleavage at or near this arginine (3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). the we used a of in epitope and radiochemical sequencing to the of Site-1 in We that cleavage not at arginine it the at leucine to be the NH2-terminal in a tetrapeptide sequence, RXXL, that as a recognition for the Site-1 We and from was from from was from and from an SREBP-2/Ras fusion protein amino of an two of the epitope novel amino acids by a sequence that of for SREBP-2 acids two novel amino acids by the sequence for acids and three of the epitope was from a X. A. Goldstein J.L. Brown M.S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), by insertion of a segment encoding between the sequences for SREBP-2 and the first of the The sequence encoding amino acids of was by Brown M.S. Goldstein J.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) with a of an site at The was with and into the site between SREBP-2 and the three of the used in of the an SREBP-2 fusion protein in which a acid region that the second transmembrane of SREBP-2 acids is replaced with the transmembrane of the LDL receptor acids Brown M.S. Goldstein J.L. Cell. Full Text PDF PubMed Scopus Google Scholar). we used mutagenesis to an in which amino acids of SREBP-2 replaced by two novel amino acids to an A of at for and at for These to amino acids of the LDL receptor by the sequence The into the site of the The an acid protein of an two of the novel amino acids SREBP-2 acids two novel amino acids LDL receptor acids two novel amino acids and SREBP-2 acids is to for the in the lumenal loop of the SREBP-2 This was by and SREBP-2 fusion proteins in which in the loop region of SREBP-2 is replaced by a novel amino acid sequence or two or by mutagenesis was with J.D. PubMed Scopus Google Scholar) the (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The confirmed by sequencing the and at two of of cells and in at in A and with (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). cells with of vector or the as (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Three after the cells to A and in the or of sterols as in the for the cells at a of (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and the cells (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The cell from was allowed to in (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) for at through a and at at for The was in of and a of protease (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google The was at for and at in a for at The is nuclear The from the was at for at in a and the was in of (1Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (854) Google Scholar) and membrane of SREBP-2 was as (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). of cells and as the cells with of vector and Three after the cells to in the of sterols as in for the cells and the cell from was The membrane was with A (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and in of A protease of the membrane in of for in the and or in the of and for after which the of was and at for as in of into the lumenal loop of sequences of the loop region of the of insertion of or two of of the membrane from cells with the for as and for at with of the and of of and of of the membrane to and with The was to for an protein that is in membranes from cells proteolytic processing of SREBP-2 with in the lumenal of nuclear of and membranes from cells with the and in the or of sterols as in the to to and with The for nuclear and membranes to for and the and cleaved NH2-terminal of The other are in cells and proteins that with the tag of the nuclear and the membrane with Scholar). was with a in proteins to membranes was with a the to the that the in and The proteins with or with a amino acids of SREBP-2 (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). with at to of cells and as the cells with of the or of of X. A. Goldstein J.L. Brown M.S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and of as the virus-associated which of G. C. Cell. Biol. PubMed Scopus Google Scholar). The cells for with and at which the was to of with and for at was and the cells with of for at The cells from and and the membrane was as The membrane from the was in of at at The was for in of and after which of of H. Goldstein J.L. Brown M.S. Cell. Full Text PDF PubMed Scopus (148) Google Scholar), and of for the was at for The was with an of of and of for and at for the of of and of SREBP-2 for of by for and at for The by with for by in for The in of Scholar) and for at at for the was to a and the with of and for at at for the was to and the was for before of the of to and to membranes the membranes to an and in a The the COOH-terminal of the cleavage was and to of an from and in a identify the position of Site-1, we a encoding a of which we SREBP-2/Ras The two of an epitope tag from the that we used (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). the terminus we amino acids of by three of an epitope from the protein of J.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of at the terminus allowed of the COOH-terminal fragment with a of the and a the COOH-terminal of SREBP-2 that SREBP-2/Ras is cleaved at Site-1 in a we cells with a vector encoding this and another encoding a in which arginine was to We used the which near levels of this protein (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). in that the 3-hydroxy-3-methylglutaryl coenzyme A reductase to cholesterol synthesis a low of to The was of sterols or it was with a of and cholesterol which cleavage at Site-1 (3Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). membranes with to and with a the terminus of SREBP-2 (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). When cells with the encoding the SREBP-2/Ras the membranes the COOH-terminal which was the of cleavage at Site-1 The of this fragment was in the of sterols The also the COOH-terminal fragment of SREBP-2 and this was also by sterols the of the cleavage of we a encoding the of SCAP, a protein that was to the cleavage of SREBPs at Site-1 to by sterols X. A. Goldstein J.L. Brown M.S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). in the of SCAP, sterols the of the COOH-terminal fragment of SREBP-2/Ras that not prevent of the cleavage of We that this is to the of cells that the which is only in cells, SREBP-2 is in cells in the of which not the increase the of the SREBP-2/Ras we which a protein that of by G. C. Cell. Biol. PubMed Scopus Google Scholar). the of the COOH-terminal fragment of the protein was by at and was still by sterols and The of the protein not to amounts of COOH-terminal cleavage in the or of the of a of the COOH-terminal fragment was was by at when with the COOH-terminal fragment and and These that Site-1 cleavage of SREBP-2/Ras of the for as the use of and the of amounts of SREBP-2/Ras to radiochemical of the of we a to use this amino acid for radiochemical that the cleavage site is located near arginine we for amino acids in the region that be replaced with methionine cleavage at two experiments in which methionine into or of the SREBP-2/Ras protein the we nuclear and with an the NH2-terminal epitope The and cleaved as from the of NH2-terminal fragment in the nucleus, and of the by sterols the in the cleaved and proteins as a are and in and is the COOH-terminal fragment be with and we cells with a encoding the SREBP-2/Ras and the cells with 35Smethionine. A membrane was with and with a of three the epitope and the COOH-terminal segment of The was to and which revealed a of that was from cells of the and the not confirmed that of the COOH-terminal fragment was the was from the membrane and used for radiochemical a of experiments in which cells with encoding the SREBP-2/Ras with the sequence in the lumenal loop or with the three methionine at and The cells with 35Smethionine. immunoprecipitation and the COOH-terminal and to and the released in was The protein with the SREBP-2 sequence not of in of the The a of in from the The and at and These that the COOH-terminal fragment with and that Site-1 cleavage between this and leucine 522 by the in the amino acid sequences of the amino acid that are for cleavage of SREBP-2 at Site-1, we a of in which was for of the amino acids in the lumenal loop between the two membrane-spanning sequences. The vector was a encoding SREBP-2 with an NH2-terminal epitope tag from the was by the which SREBP-2 to not so as to the for proteolysis (2Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). cells in or after which nuclear and membrane to and with an the NH2-terminal epitope from of the scanning When the the SREBP-2 sequence, we the NH2-terminal fragment of SREBP-2 in nuclear This fragment was in the of sterols The was not cleaved the and cleaved as as the protein and the of scanning the we the sequences of the lumenal of of the SREBPs that two from the (5Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 6Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (501) Google Scholar) and two from the R. J. Wang X. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). two cleavage the and the and Three of the cleavage and of these the The other not cleavage. These the of to Cleavage was also not affected when the the cleavage site was to These that arginine at the P4 position and leucine 522 at the position are the only in the region of Site-1 that the for arginine we replaced this with When was for this only a of cleavage was acid and a of cleavage. other cleavage. The of arginine was Cleavage was when we replaced arginine with and a arginine at or These the position of the arginine the NH2 terminus or We also the of the for leucine 522 at the cleavage site Cleavage was not when this was to or It was when leucine 522 was to acid or The at the position was not This is in and it is acid and in and of in SREBP-2 with acid or cleavage. The cleavage was also when this was to or A was when it was to the second transmembrane to recognition by the Site-1 we replaced this sequence with the membrane-spanning region of another the LDL receptor The LDL receptor is a transmembrane protein of the membrane with a membrane-spanning segment oriented with NH2 terminus in the and terminus in the Brown M.S. Goldstein J.L. Cell. Full Text PDF PubMed Scopus Google Scholar). This is the as the of the second transmembrane of SREBP-2 (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The protein the LDL receptor transmembrane was cleaved as as the as by the of NH2-terminal fragment in the nucleus Cleavage was by sterols It was also when arginine of the was to These that the sequence of the second transmembrane is not for cleavage at Site-1 A sequence at this position is When the second transmembrane was replacement by another transmembrane cleavage at Site-1 and was not we that the lumenal loop sequence is in the ER lumen (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This was of a encoding a protein that an segment amino into the lumenal loop (4Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). We to a protein segment because not by after insertion into the lumenal loop of membrane from cells this and the segment the epitope was to be from by in the not the of The segment was also to The with these is that the protein was not cleaved by the Site-1 we not be that the not the of the this we of the that the sequence the NH2-terminal of arginine is not for Site-1 cleavage. we a encoding of SREBP-2 in which we or amino acids into this sequence in of The amino acids or two for membrane the SREBP-2 with and the in was by As in the of the SREBP-2 with or two was by with and and by and not by and The for the with two This that the lumenal loop sequence of the The of processing to an that the is located in the ER and not the of nuclear and membrane from cells SREBP-2 with the lumenal sequence, the or the insertion of or two The cells that SREBP-2 cleavage or cleavage The with site and the protein and and the with two the nuclear the NH2-terminal of proteins the Cleavage of proteins was by This that the of SREBP-2 to sterol-regulated cleavage at Site-1 and cleavage at The of the cleavage. The of cleavage was by for the site and by for the protein two the in this that the Site-1 protease cleaves the between leucine 522 and in the lumenal loop of The only that to be for recognition are arginine and leucine The of the arginine to the leucine also to be The recognition sequence to be where can be or at The sequence is in the and SREBPs in and also in from the J.B. Spiegelman B.M. Cell. Biol. 1993; PubMed Scopus Google Scholar) and U. S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). We that other of the lumenal loop are also for cleavage because the sequence to other in the lumenal loop cleavage not the identify the site within SREBP-2 that is cleaved by the Site-1 not where in the cell this cleavage R. J. Wang X. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and cell A. S. and J. that SREBPs are membranes of the nuclear envelope and We not the Site-1 protease in these or the SREBPs be transported to other site where cleavage We and for for with and for synthesis of and and and for with radiochemical amino acid sequence