Overexpression of Membrane Domain of SCAP Prevents Sterols from Inhibiting SCAP·SREBP Exit from Endoplasmic Reticulum

Abstract

SCAP (SREBP cleavage-activating protein) forms a complex with sterol regulatory element-binding proteins (SREBPs) and escorts them from the endoplasmic reticulum (ER) to the Golgi complex where proteases release transcriptionally active segments of SREBPs, which enter the nucleus to activate lipid synthesis. The NH2-terminal segment of SCAP contains eight transmembrane helices, five of which (TM2–6) comprise the sterol-sensing domain. This domain responds to sterols by causing the SCAP·SREBP complex to be retained in the ER, preventing proteolytic release and reducing transcription of lipogenic genes. Here, we use transfection techniques to overexpress a segment of SCAP containing transmembrane helices 1–6 in hamster and human cells. This segment does not interfere with SCAP·SREBP movement to the Golgi in the absence of sterols, but it prevents sterols from suppressing this movement. This block is abolished when SCAP(TM1–6) contains a point mutation (Y298C) that is known to abolish the activity of the sterol-sensing domain. We interpret these findings to indicate that sterols cause the SCAP·SREBP complex to bind to an ER retention protein through an interaction that involves the sterol-sensing domain. The SCAP(TM1–6) segment competes with the SCAP·SREBP complex for binding to this putative retention protein, thereby liberating the SCAP·SREBP complex so that it can move to the Golgi despite the presence of sterols. These studies provide a potential mechanistic explanation for the ability of sterols to block SCAP·SREBP movement from the ER and thereby to control lipid synthesis in animal cells. SCAP (SREBP cleavage-activating protein) forms a complex with sterol regulatory element-binding proteins (SREBPs) and escorts them from the endoplasmic reticulum (ER) to the Golgi complex where proteases release transcriptionally active segments of SREBPs, which enter the nucleus to activate lipid synthesis. The NH2-terminal segment of SCAP contains eight transmembrane helices, five of which (TM2–6) comprise the sterol-sensing domain. This domain responds to sterols by causing the SCAP·SREBP complex to be retained in the ER, preventing proteolytic release and reducing transcription of lipogenic genes. Here, we use transfection techniques to overexpress a segment of SCAP containing transmembrane helices 1–6 in hamster and human cells. This segment does not interfere with SCAP·SREBP movement to the Golgi in the absence of sterols, but it prevents sterols from suppressing this movement. This block is abolished when SCAP(TM1–6) contains a point mutation (Y298C) that is known to abolish the activity of the sterol-sensing domain. We interpret these findings to indicate that sterols cause the SCAP·SREBP complex to bind to an ER retention protein through an interaction that involves the sterol-sensing domain. The SCAP(TM1–6) segment competes with the SCAP·SREBP complex for binding to this putative retention protein, thereby liberating the SCAP·SREBP complex so that it can move to the Golgi despite the presence of sterols. These studies provide a potential mechanistic explanation for the ability of sterols to block SCAP·SREBP movement from the ER and thereby to control lipid synthesis in animal cells. sterol regulatory element-binding protein SREBP cleavage-activating protein N-acetyl-leucinal-leucinal-norleucinal cytomegalovirus endoglycosidase H endoplasmic reticulum human embryonic kidney 293 cells herpes simplex virus polymerase chain reaction transmembrane helices 1–6 Xpress epitope tag kilobase(s) polyacrylamide gel electrophoresis The gated movement of sterol regulatory element-binding proteins (SREBPs)1 from endoplasmic reticulum (ER) to the Golgi complex controls the rate of lipid synthesis in animal cells (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2999) Google Scholar). SREBPs are membrane-bound transcription factors whose NH2-terminal domains must be released proteolytically to enter the nucleus. For proteolysis to take place, the SREBPs must be transported from their site of synthesis in the ER to their site of proteolysis in the Golgi complex (2DeBose-Boyd R.A. Brown M.S. Li W.-P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). This transport is accomplished by SCAP (SREBP cleavage-activating protein), a membrane-bound protein that forms a complex with SREBPs in the ER and escorts them to the Golgi complex. SREBPs are tripartite membrane proteins of ∼1150 amino acids in length (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2999) Google Scholar). The NH2-terminal domain of ∼480 amino acids contains a basic-helix-loop-helix-leucine-zipper motif that allows it to bind DNA and activate transcription. This domain is followed by a membrane attachment domain of ∼80 amino acids consisting of two transmembrane helices separated by a short hydrophilic loop that projects into the lumen of the ER. The COOH-terminal domain of ∼590 amino acids performs a regulatory function. The SREBPs are oriented in the membrane in a hairpin fashion with the NH2- and COOH-terminal domains projecting into the cytoplasm (3Hua 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). SCAP is a polytopic membrane protein with two distinct domains (4Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The NH2-terminal domain of ∼550 amino acids consists of alternating hydrophobic and hydrophilic sequences that are believed to form eight membrane-spanning helices. The COOH-terminal domain of ∼725 amino acids is hydrophilic and projects into the cytoplasm. This domain contains five copies of a WD-40 repeat sequence that is found in many proteins and mediates protein/protein interactions (5Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). Indeed, immediately after its synthesis, SCAP forms a complex with SREBPs that is mediated by an interaction of the WD-40 repeat domain of SCAP and the COOH-terminal regulatory domain of the SREBPs (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Sakai 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). When cells are depleted of sterols, the SCAP·SREBP complex travels from the ER to the Golgi, where it encounters two proteases that act in sequence to release the NH2-terminal domain of SREBP into the cytoplasm (2DeBose-Boyd R.A. Brown M.S. Li W.-P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 8Nohturfft A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar). The first enzyme, designated Site-1 protease, cleaves the SREBP in the luminal loop, thereby separating the two transmembrane helices (9Sakai 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 (325) Google Scholar). This allows Site-2 protease to cleave the first transmembrane helix within the plane of the membrane, releasing the NH2-terminal domain with three hydrophobic amino acids attached (10Duncan E.A. Davé U.P. Sakai J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1998; 273: 17801-17809Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This domain travels to the nucleus where it activates transcription of multiple genes involved in the synthesis of cholesterol and fatty acids as well as their uptake from plasma lipoproteins through the low density lipoprotein receptor (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2999) Google Scholar). Movement of SREBP to the Golgi absolutely requires SCAP. In SCAP-deficient mutant cell lines, the SREBPs fail to move to the Golgi complex, and they are not released proteolytically from membranes. As a result, SCAP-deficient cells are cholesterol auxotrophs (11Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The SCAP·SREBP transport process is controlled by cholesterol. When sterols build up in cells, the SCAP·SREBP complex fails to bud from ER membranes and it never reaches the Golgi. 2Nohturfft, A., Yabe, D., Goldstein, J. L., Brown, M. S., and Espenshade, P. J. (2000) Cell 102, 315–323. As a result, the synthesis of cholesterol and fatty acids is reduced. This sterol-regulated transport mechanism modulates the cholesterol content of cell membranes. The sterol-sensing function in the SREBP system has been traced to a portion of the membrane domain of SCAP encompassing transmembrane helices 1–6 (TM1–6). This region contains sequences that are shared with three other proteins that are postulated to interact with sterols (12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). This segment has therefore been termed “the sterol-sensing domain” (13Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). Point mutations at two conserved residues within this domain (Y298C and D443N) abolish the sterol-sensing function of SCAP (12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar, 14Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). These mutant forms of SCAP form complexes with SREBPs that are transported to the Golgi normally, but they can no longer be blocked by sterols.2 As a result, cells bearing these SCAP mutations overproduce cholesterol and fail to shut-off cholesterol synthesis when intracellular sterol levels rise. A crucial question relates to the mechanism by which SCAP is retained in the ER in sterol-overloaded cells. Two mechanisms must be considered. The first model postulates that the SCAP·SREBP complex leaves the ER constitutively and that sterols cause the complex to bind to a molecule, presumably a protein, that actively retains it in the ER. The alternative hypothesis states that ER retention of the complex represents the constitutive state. For the SCAP·SREBP complex to leave the ER, it must interact with a protein that carries it to the Golgi. This interaction would occur only in sterol-depleted cells. The current studies were designed to distinguish between these two possibilities. For this purpose, we transfected cells with cDNAs encoding a portion of the transmembrane domain of SCAP that includes the sterol sensor. Overexpression of this domain abolished the ability of sterols to suppress SCAP movement and SREBP cleavage. This effect was not observed when the transmembrane domain contained the Y298C mutation. These findings support the model in which the SCAP·SREBP complex is retained in the ER through an interaction between the transmembrane domain and an unidentified retention protein. The overexpressed transmembrane domain competes for this interaction and allows the SCAP·SREBP complex to move constitutively even in the presence of sterols. We obtained monoclonal HSV-Tag antibody (IgG1) from Novagen; monoclonal anti-Xpress antibody (IgG1) from Invitrogen; rat anti-mouse horseradish peroxidase-conjugated antibody (κ-chain) from ICN; 2-hydroxypropyl-β-cyclodextrin from Cyclodextrin Technologies Development, Inc.; and trypsin (catalog number LS003744) from Worthington Biochemical Co. All other reagents were obtained from sources as described previously (7Sakai 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, 11Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). Lipoprotein-deficient serum (d > 1.215 g/ml) was prepared by ultracentrifugation (15Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Expression vectors pTK, pTK-HSV-BP2, pTK-HSV-BP2(R519A), pTK3-SCAP, and pCMV-SCAP have been previously described (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar, 16Hua 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). All expression vectors described below were driven by the cytomegalovirus (CMV) promoter-enhancer contained in the pcDNA3.1HisC vector or pcDNA3 vector (Invitrogen). pCMV-Xp-SCAP(1–448) was amplified by PCR of by of at with a of which amino acids by a sequence and and which amino acids followed by a and The PCR was and and the was and into the of pcDNA3.1HisC vector to the was amplified by PCR of by A. with the of and in the was amplified by PCR of pCMV-SCAP with a of which amino acids by an and a and which amino acids followed by a and an The PCR was into pcDNA3 vector in a fashion as described was amplified by PCR of pCMV-SCAP with a of encoding amino acids by a and encoding amino acids followed by a and a and were amplified with and of the encoding amino acids followed by a and encoding amino acids followed by a and encoding amino acids followed by a and The PCR were into pcDNA3.1HisC vector as described was by by A. with and and the was and into of the pcDNA3 was by by A. and and the was and into the of to the The was with and the was and into pcDNA3 vector as described vectors of 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 pcDNA3 or of human embryonic kidney cells were up at and in at in A containing and with the cells were transfected with the as described previously (3Hua 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). after the cells were to A containing and in the absence or presence of sterols of cholesterol in a of as in the for or N-acetyl-leucinal-leucinal-norleucinal at a of was to for cells from two were and for and membrane as described previously J. E.A. Rawson R.B. X. Brown M.S. Goldstein J.L. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of cells, a of mutant hamster cells that SCAP (11Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (149) Google were up and in at in of and containing and with and the cells were transfected with the as described previously (11Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). or the cells were with and with or containing and containing the as described in the to and of NH2- and COOH-terminal segments of SCAP in transfected SCAP-deficient hamster cells. cells were up for as described in The in the transfection were as of of pCMV-Xp-SCAP(1–448) and of and and of The of DNA was to by of vector pcDNA3.1HisC after the cells were for in in the absence and or presence and of cholesterol was for and the cells were and The membrane was and to as described of antibody The and from the of two of cells were to and with or of monoclonal followed by a of rat anti-mouse horseradish peroxidase-conjugated antibody the NH2-terminal portion of SCAP containing the transmembrane segments the COOH-terminal portion of SCAP containing the transmembrane segments and and the COOH-terminal domain. The were to for of cells were and membrane were prepared and with trypsin and endoglycosidase H as described previously A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google that the H was for at Cell were and membrane were prepared as described previously (12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). The membrane were with of as described (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google that the was through a five and for at was as described (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). and were as described the system (7Sakai 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). Two SCAP were a monoclonal antibody hamster SCAP acids (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google and a antibody hamster SCAP acids and (6Sakai J. Nohturfft A. Cheng D. Ho Y.K. Brown M.S. Goldstein J.L. J. Biol. Chem. 1997; 272: 20213-20221Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). a of the membrane of the two segments of SCAP that were by the cDNAs in these The of the the three and the Y298C mutation are The sterol-sensing domain of SCAP transmembrane helices in an in which human cells were transfected with a encoding with an epitope tag at the The cells were transfected with cDNAs encoding a portion of the membrane domain of SCAP helices with an Xpress The protein contained the sequence or the Y298C mutation. The cells were in the absence of sterols or in the presence of of a of and after which and membrane were to gel electrophoresis and with the or the the SCAP. When the cells were in the absence of sterols, the SREBP was and the NH2-terminal segment was found in the The of sterols abolished and the segment Expression of the not SREBP in the absence of sterols but it of by sterols When the protein the Y298C it the ability to block sterol The of that the and SCAP proteins were at levels in the cells. The of that the of sterol the first transmembrane helices of SCAP. of cDNAs encoding segments that transmembrane segments to abolish sterol and was abolished only when the contained transmembrane domains 1–6 and In the of we transfected cells with a encoding a form of SCAP that from amino acids to This eight transmembrane helices the of the WD-40 domain it is at a that is amino acids from the of the protein. studies have that this protein does not interact with SREBPs as by a of does it SREBP to SCAP-deficient cells. J. Goldstein, and M. S. Brown, when we transfected the cells with sterols When we sterol was abolished The effect of this SCAP was when we transmembrane helices the that these transmembrane helices were for this The of that of the and the transmembrane were in the cells. The membrane domain of SCAP contains three that are attached to that into the ER lumen (4Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In sterol-depleted cells, SCAP to the Golgi complex where the are by that them to by H A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar). When cells are in the presence of sterols, the SCAP·SREBP complex is in the ER, and the in their state. the in H we first membrane with trypsin to the of the thereby between and forms A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar, 12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). of the contains two of the and this can be by with an antibody an epitope this an designed to the of SCAP in cells that were transfected with cDNAs encoding SCAP. This was does not the segment that is and it does not with the antibody the the antibody the from SCAP. When the cells were transfected only with and in the absence of sterols, the SCAP retained or two after with that the were When the cells were in the presence of sterols, the SCAP were to H and the protein at a to When we the encoding was no effect SCAP in the absence of sterols in the presence of sterols the SCAP that of the SCAP to to the Golgi complex. When contained the Y298C it no effect the of SCAP in the absence or presence of sterols and and This that the segment prevents sterols from the movement of SCAP from ER to Golgi. The of support the hypothesis that the segment with a putative protein that retains SCAP in the ER in the presence of sterols. This interaction the SCAP·SREBP complex from the protein, thereby it to to the Golgi even in the presence of sterols. The of was to SCAP activity be in SCAP-deficient cells by of SCAP(TM1–6) a encoding the of the protein When the cells were transfected with a encoding in the absence of was no to the form of the segment not transfection of a encoding the NH2-terminal segment with the sequence or the Y298C mutation was only when we with containing the sequence or the Y298C mutation The of this effect was by the that no of SCAP segments of the mutant of which is not by Site-1 protease (9Sakai 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 (325) Google 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 two of that the segments of SCAP were in the transfected cells as by with the which the NH2-terminal or which the COOH-terminal The in that the NH2- and COOH-terminal segments of SCAP are to interact so as to SCAP activity even they are as this interaction we a SCAP-deficient cells were transfected with cDNAs encoding the COOH-terminal segment of with the NH2-terminal segment containing the or Y298C were with which only the NH2-terminal The and from the were to and with or with which the COOH-terminal As in the of a of the COOH-terminal segment was found in the after with the antibody the NH2-terminal segment that these two segments were The was the in the absence and presence of sterols. the NH2-terminal and COOH-terminal segments of SCAP can interact when transfected into cells, they be to the of SCAP-deficient cells, which are for cholesterol (11Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The of in which cells were transfected with SCAP segments and for in the absence of that this was the The current that of a portion of the transmembrane domain of SCAP helices the ability of sterols to cause the retention of the SCAP·SREBP complex in the ER. the SCAP·SREBP complex to the Golgi complex where the of SCAP are to an form and where SREBP is by the Site-1 and Site-2 proteases to the transcriptionally active NH2-terminal When the SCAP(TM1–6) segment contains the Y298C its ability to block sterol of SCAP·SREBP movement is reduced. The hypothesis to these postulates that the SCAP(TM1–6) segment competes with the SCAP·SREBP complex for binding to a protein that retains the complex in the ER in the presence of sterols. This hypothesis is by the that the Y298C mutation in the SCAP(TM1–6) segment this The Y298C mutation in SCAP has been to the protein to sterols (12Nohturfft A. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12848-12853Crossref PubMed Scopus (111) Google Scholar). does not for the retention protein of SCAP is to bind sterols, which in binding to the retention protein, to bind sterols, but it bind to the retention protein. of these mechanisms would the of to be by sterols as well as the that does not with SCAP·SREBP for the retention protein in the presence of sterols. SCAP(TM1–6) includes the sterol-sensing domain (TM2–6) that is shared with three other proteins in M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). be to the sterol-sensing domains in these proteins interact with the protein that retains SCAP and SCAP(TM1–6) interfere with the of these of these proteins is A an of cholesterol that is to ER membranes X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). the of this in an that requires the sterol-sensing domain Goldstein J.L. Brown M.S. Cell. Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). We are to of SCAP(TM1–6) interfere with the ability of sterols to the of A in these studies is that the NH2-terminal and COOH-terminal domains of can the SREBP transport function of SCAP in SCAP-deficient hamster cells. This was to the ability of these two segments to form a complex that was to The COOH-terminal segment that we contains three distinct segments and the loop that these and the or of these must be of binding to the thereby the function of SCAP. the mechanism of SCAP(TM1–6) activity is this protein segment provide a for the postulated ER retention protein with which it We Nohturfft for SCAP and of the for and for with and and for synthesis and DNA