Key points are not available for this paper at this time.
When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles. When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles. How to Block Cholesterol Synthesis Journal of Biological ChemistryVol. 280Issue 28PreviewWhen added to living cells, sterols can block the synthesis of cholesterol and other lipids by preventing several membrane-embedded transcription factors from being transported to the Golgi complex where they are activated. These sterol regulatory element-binding proteins (SREBPs) are transported from the endoplasmic reticulum (ER) to the Golgi in a process that requires them to form complexes with the escort protein Scap. Subsequent clustering of the Scap/SREBP complex into transport vesicles involves binding to the coat proteins Sec23/24, which is mediated by the small GTPase Sar1. Full-Text PDF Open Access In mammalian cells the synthesis of cholesterol and other lipids is governed by the lateral movement of a membrane-embedded protein complex into coated vesicles that bud from the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CMV, cytomegalovirus; GST, glutathione S-transferase; SREBP, sterol regulatory element-binding protein; TK, thymidine kinase; VSV, vesicular stomatitis virus; CHO, Chinese hamster ovary; HSV, herpes simplex virus; GMP-PNP, guanyl-5′-yl imidodiphosphate. and move to the Golgi. This membrane sorting event is blocked selectively by cholesterol and related oxysterols, and it constitutes the only known instance in which ER-to-Golgi transport of a protein is regulated selectively by a small molecule (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). An understanding of this regulatory mechanism is therefore essential to a general understanding of regulated ER-to-Golgi transport as well as to an understanding of lipid metabolism. The crucial components of the sterol-regulated protein complex are sterol regulatory element-binding proteins (SREBPs), which are membrane-embedded transcription factors that are synthesized on ER membranes (3Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). SREBPs are anchored to ER membranes by virtue of two transmembrane helices. To activate lipid synthesis, the SREBPs must be transported to the Golgi where proteases release the active domains that enter the nucleus. ER-to-Golgi transport requires that the SREBPs form complexes with Scap, an escort protein that contains eight membrane-spanning helices (4Sakai 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). In sterol-depleted cells, the Scap/SREBP complex clusters laterally with other proteins into COPII-coated vesicles that bud from the ER (5Nohturfft 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). Like other proteins that cluster into COPII-coated vesicles (6Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Biol. 2004; 20: 87-123Crossref Scopus (706) Google Scholar), clustering of the Scap/SREBP complex is initiated by Sar1, a small GTPase that binds to ER membranes in a GTP-dependent fashion (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). Sar1 recruits a heterodimeric protein called Sec23/24, and this recruits another heterodimer, Sec13/31, which forms the coat of the COPII coated vesicle, thereby permitting vesicle budding. Using in vitro binding assays with purified COPII components, Espenshade et al. (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar) demonstrated that Sec23/24 binds to the Scap/SREBP complex in a fashion dependent upon Sar1-GTP. This binding did not occur when membranes were isolated from intact cells that were preincubated with sterols, a condition in which the Scap/SREBP complex does not move to the Golgi (5Nohturfft 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). To gain a molecular understanding of the mechanism by which sterols block the binding of COPII proteins to the Scap/SREBP complex, it is necessary to establish an in vitro system in which binding can be blocked by addition of sterols to isolated membranes, rather than to preincubated cells. The feasibility of such an assay is increased by the recognition that sterol-mediated inhibition of Scap/SREBP transport requires Insigs, which are resident proteins of the ER that have an anchor function. Addition of sterols to intact cells or to isolated membranes triggers the binding of Scap to Insigs, an event that is required for sterol-mediated inhibition of Scap/SREBP transport (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar). The Insig requirement when the membrane of Scap in cells by the and sterols blocked ER-to-Golgi transport (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). This by of or (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). The Insig requirement when cells were to be to sterol-mediated inhibition of Scap/SREBP transport DeBose-Boyd R.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we that sterols can block the Sar1-dependent binding of COPII proteins to the Scap/SREBP complex when added to isolated membranes in vitro and that this inhibition requires we a hexapeptide sequence in a cytoplasmic loop of Scap that is required for COPII binding in vitro and for ER-to-Golgi movement of the Scap/SREBP complex in intact cells. These studies that membranes of the components required for sterol-mediated of Scap/SREBP transport and thereby for of lipid synthesis in cells. from GMP-PNP, and from and from and from and from and protein from of and were as M.S. Goldstein J.L. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Brown M.S. Goldstein J.L. J. PubMed Scopus Google Scholar). by J.L. Brown M.S. PubMed Scopus Google Scholar). and from from from and and from The were in the hamster Scap and (4Sakai 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), hamster Scap (4Sakai 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), D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar), and D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). were in the which of by of a of the (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google which with an of the thymidine (4Sakai 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 which the of hamster Scap (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google and which of protein from the of vesicular stomatitis by two of a of the (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). The were by J. and which hamster Scap and by of a of the and which hamster Scap were with P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar) as the and which hamster Scap and by of a of the and which hamster Scap were with as the which by of the with as the of were with the were contains a of and with and contains with and contains with and Cell and cells, a from cells DeBose-Boyd R.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), were in in in with and cells were for in of the a of cells cells were with in the to and in of with as a DeBose-Boyd R.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of to by addition of the cells were with for in of were with and with of for to for use in the assays of COPII proteins to by in the presence of cells were for as on cells were with of of and the of on cells were with of of or the of and of and the cells were to for the cells were with in the presence of the of 25-hydroxycholesterol the cells were and the microsomal membranes were for Scap binding to COPII membranes were with of heterodimeric complex and of in the presence of for the microsomal membranes were by with and with The and were to and with and as of were to for and vitro addition of sterols COPII proteins binding to Scap in membrane vesicles. cells were for as cells were with of of and of in the or presence of of the cells were with for as in the to The cells were to and for The cells were for of microsomal of the membranes were in a of of the of the sterols added in of and or of and on a for the for and the in and for Scap binding to COPII proteins as in with the and were to and with and of were to for and of Scap transmembrane on binding COPII binding and of on cells were for as cells were with of and of and as cells were with for The cells were to in the or presence of 25-hydroxycholesterol for the in and with of as The and from the were to and with and Sar1 cells were with of of and of in the presence or of of as were in the as for A. were for assay of Scap binding to COPII proteins as in the to protein complexes were isolated by and the and were to and with and cells were with of of and the of or cells were with for as in The cells were to and for The cells were for as T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which of the membrane and were to and with and were to for of in Scap cytoplasmic loop that are required for and COPII binding of for of in the loop of in which or by and were to for the and not and were and were from and on cells were for as cells were with of and of or as in cells were with for The cells were to for cells were and to Sar1 cells were and as for were for assay of Scap binding to COPII proteins as in the to protein complexes were isolated by and the and were to and with and were to for in loop of Scap sorting of Scap into COPII vesicles. and on cells were for as cells were with of of or and of cells were with for The cells were to in the or presence of 25-hydroxycholesterol for the in and with of as The and from the were to and with and in vitro cells were with of of and of or or of or as were in the as for A. were with in the presence of for and for as The vesicle and membrane were to and with of membrane and were to for hamster and were in as proteins (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). is a heterodimeric complex of with two and with a (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). These proteins were in cells, and the purified by as (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar) with and were through a with with and and purified proteins were in and for to of of membranes from cells were isolated as (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) with the were in membranes were and in with for of COPII to assay as (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar) with several in a of of of microsomal membrane of of of Sec23/24 complex, and were for were by for The in of and and with for for the with for to The were by of the with of and to and The with which proteins were by with of for by An of the to and for Scap or of cells P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar) were in for in (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar). the cells were the were in and a from of cells on and in of with and were The for which the for and for In the to the The as into of in and In from by the of Schekman Schekman J. Cell Biol. PubMed Scopus Google Scholar) and T. PubMed Scopus Google Scholar) and as (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). in a of of of microsomal membranes from cells, of and for the membrane and the vesicle were by and to and as (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). from two of cells were in of in and through a eight by for the by for the to as (7Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar), that the of and were and were with molecular and were used the and and were used a of were by the system and to for the demonstrated complex Scap and the COPII Sec23/24 (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). of this complex is initiated by Sar1 in the of Sar1 to Scap requires the Sec23/24 complex (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). In the we used the pull-down assay that used by Espenshade et al. (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). cells were with of Scap, and were in to of the 25-hydroxycholesterol to the cells were and microsomal membranes were in vitro with a mammalian or Sar1 and Sec23/24 for the membranes were with with and as The proteins were to and with to Scap or to the on Sar1 or of the in the of the on the glutathione When a of Sec23 when the were in the presence of and not in the presence of and This is with that the complex is by (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). of the cells with 25-hydroxycholesterol on the pull-down of that binding of Sar1 to Sec23/24 is not regulated by In the presence of GMP-PNP, Scap with the complex this did not occur when the cells were with 25-hydroxycholesterol When the of Sar1 Sec23 and Scap were in the presence of or of cells with 25-hydroxycholesterol the binding of Scap to the These the of Espenshade et al. (2Espenshade P.J. Li W.-P. Yabe D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11694-11699Crossref PubMed Scopus (123) Google Scholar). In cells, the sterol-mediated inhibition of Scap from the ER requires Insig proteins T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). When Scap is by of a by the and sterols to Scap Insig protein is by T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To the sterol-mediated inhibition of COPII binding to Scap requires we the pull-down assay in cells that Scap or Scap two of In the of with sterols did not prevent Scap binding to the COPII complex and of of inhibition of this binding with the COPII complex a with the that Insig proteins not the ER T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). The of Scap a binding for T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar), and it to the Golgi in the presence of 25-hydroxycholesterol (5Nohturfft 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). with to to COPII proteins in cells that and were with 25-hydroxycholesterol In the 25-hydroxycholesterol binding of COPII proteins to Scap We to sterols Scap binding to COPII proteins when added to microsomal membranes in vitro rather than to the intact cells a for we cells with a a transmembrane protein that the ER by binding to COPII proteins T. J. Cell Biol. PubMed Scopus Google Scholar). When membranes were from cells Scap in the of Insig-1, Scap with and sterols did not this binding when added in in or in the presence of as a were with protein When the cells Insig-1, the in vitro addition of 25-hydroxycholesterol or in binding to Scap not and which does not block movement of the Scap/SREBP complex to the did not block the with Scap, when added Cholesterol selectively blocked binding of to Scap not to of in the cholesterol added as a complex with as a To the of Scap responsible for binding COPII we the membrane-spanning of Scap with on the cytoplasmic loop transmembrane helices and we to this loop as loop This is the of on the cytoplasmic of Scap, the two and To explore the of this we Scap with and them into cells. loop contains the loop the membrane-spanning forms of Scap were when in cells, and to in a fashion as by with In the of to that with and These that loop is not required for sterol of Scap binding to To loop is required for Scap binding to COPII we the isolated membranes, and the pull-down assay In the of Insig-1, and were with and 25-hydroxycholesterol did not this and When 25-hydroxycholesterol the of forms of Scap with and the other not by condition These that loop is required for Scap to with the COPII protein To which of loop is required for the Sar1 we the protein the of the loop To the of this we a it with a transmembrane helices and the of Scap in studies that when a the of Scap is with a the of Scap, the two of Scap a thereby sterol-regulated transport of SREBPs from ER to Golgi T. Goldstein J.L. Brown M.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). when with the processing of as by an in in this These that the in loop of Scap are required for COPII binding and transport to the Golgi. To the loop that are required for COPII we a of in of in this loop We the into cells by with a and the of the of by These that and to a in These a hexapeptide which and the that by the of the other loop and the of Scap to to COPII proteins as by the pull-down assay in vitro which the sequence to the Scap binding to COPII proteins this did not processing in intact cells is that the by the pull-down assay a of this protein for the of the in vitro assay that the of Sar1 the is to this thereby permitting of the complex from the To explore the of the loop of Scap, we the of of to to in a fashion cells were with or Scap, Insig-1, and When the cells were preincubated with Scap and were with The that Scap binds and of Scap to regulated by that this the to sterol The to to COPII proteins is a in this protein. assay to Scap is the in vitro assay (1Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). membranes are isolated from cells in the or presence of The membranes are in vitro with which the of COPII-coated vesicles. The vesicles are from the membrane by and the Scap of the vesicles is by and The of with membranes from cells that with Scap or and Insig-1, which is necessary for sterol In the of sterols, Scap in the vesicle and this when the cells were with 25-hydroxycholesterol not in the vesicle in the presence or of 25-hydroxycholesterol and We the cells with a a of Scap that the The of this protein into the vesicle and this when the cells were with 25-hydroxycholesterol When the to the vesicle and and biochemical studies in and mammalian cells have a of protein binding that to the sorting of ER membrane proteins into COPII-coated vesicles that move to the Golgi complex in M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Biol. 2004; 20: 87-123Crossref Scopus (706) Google Scholar, Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, and Cell Biol. Full Text PDF PubMed Scopus (123) Google Scholar). is into the mechanism by which this sorting be regulated by small In the we as to establish an in vitro system in which this can be Using the Scap/SREBP transport process as a we show that addition of 25-hydroxycholesterol to isolated membranes in vitro blocks the Sar1-dependent binding of the Sec23/24 complex to Scap. This block on the presence of the Insig anchor proteins in the ER We show that binding of Sec23/24 to Scap requires a hexapeptide in the cytoplasmic loop the and transmembrane helices of Scap. The mechanism by which sterols to block Sec23/24 binding to Scap to be studies have that cholesterol binds to Scap A. Brown M.S. Goldstein J.L. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and J. Brown M.S. Goldstein J.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), thereby Scap to to Scap to to Insigs, it does not to Scap A. Brown M.S. Goldstein J.L. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), does it a J. Brown M.S. Goldstein J.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We that 25-hydroxycholesterol acts through another ER protein that is to be When Scap binds to Insigs, the COPII proteins to Scap. does not in the Scap loop binds to Insig in a fashion the Insig the hexapeptide in loop it to complex to be the of other ER proteins in this regulatory The general for Sec23/24 binding to Scap to the for other ER with other proteins J. J. Cell Biol. PubMed Scopus Google Scholar, Orci L. T. S. Schekman R. Cell. Full Text PDF PubMed Scopus Google Scholar), we that forms a complex with from general in transport of the complex forms of with Scap In sterol-depleted membranes this complex binds to Scap. with other proteins for COPII coated vesicles D. S. Orci L. Schekman R. Cell Biol. PubMed Scopus Google Scholar), this binding to be when Sar1 to In of to be the that binds to the protein (6Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Biol. 2004; 20: 87-123Crossref Scopus (706) Google Scholar, Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Cell Biol. PubMed Scopus Google Scholar). We have to that this is when Scap is the The only in the is that Scap binding to Sec23/24 is when cholesterol Scap to to We not is in binding to Scap or is to Sec23/24 to the The of the MELADL hexapeptide to be The is that Sec23/24 binds to this of on proteins have to be binding for the Sec23/24 complex Cell Biol. Full Text Full Text PDF PubMed Scopus Google and Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). are as is the MELADL of two as in two the two in studies that two binding for proteins Goldberg J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of binds the sequence in the protein of the the MELADL sequence in Scap. studies with that the and the of the MELADL sequence be required for of Scap studies be necessary to the of the MELADL sequence that are responsible for sterol-regulated Sec23/24 binding and into COPII vesicles. We and DeBose-Boyd for and with
Sun et al. (Tue,) studied this question.