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• W2005218056 abstract "Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from ∼5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterol-sensing domains in SCAP and HMG CoA reductase. Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from ∼5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterol-sensing domains in SCAP and HMG CoA reductase. Insig-1 and Insig-2 were recently identified as membrane proteins that reside in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; endo H, endoglycosidase H; HEK, human embryonic fibroblasts; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; PNGase F, peptide N-glycosidase F; PBS, phosphate-buffered saline; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; TK, thymidine kinase. 1The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; endo H, endoglycosidase H; HEK, human embryonic fibroblasts; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; PNGase F, peptide N-glycosidase F; PBS, phosphate-buffered saline; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; TK, thymidine kinase. and play a central role in the regulation of cholesterol and fatty acid synthesis (1Yang 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 (768) Google Scholar, 2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar, 3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 4Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). When cellular cholesterol levels are elevated, Insigs bind to SCAP and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) through interactions with the sequence-related sterol-sensing domains in the membrane-spanning regions of each protein. In the case of SCAP, Insig binding prevents movement of the SREBP/SCAP complex from the ER to the Golgi, thus blocking proteolytic cleavage and transcriptional activation of SREBP (1Yang 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 (768) Google Scholar, 2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar). In the case of HMG CoA reductase, Insig binding promotes ubiquitination and degradation, thus slowing the rate-limiting enzymatic reaction in cholesterol synthesis (3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 4Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar).SREBPs are a family of membrane-bound transcription factors that control the synthesis of cholesterol and fatty acids in animal cells (5Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1093) Google Scholar). The C terminus of SREBP binds to the C terminus of SCAP, the protein that transports SREBPs from the ER to the Golgi when cells are depleted of sterols. Within the Golgi, two resident proteases cleave SREBP through regulated intramembrane proteolysis (Rip), releasing the N terminus of SREBP from the membrane and allowing it to translocate to the nucleus and activate transcription of target genes (6Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar).In the presence of sterols, SCAP undergoes a conformational change, causing the SCAP/SREBP complex to be retained in the ER and thus abrogating SREBP-dependent transcription. Insig proteins have been found to facilitate both the ER retention of the SCAP/SREBP complex (1Yang 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 (768) Google Scholar, 2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar) and the sterol-sensitive SCAP conformational change (7Adams C.M. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10647-10652Crossref PubMed Scopus (89) Google Scholar). These activities of Insig occur via an interaction with the sterol-sensing domain of SCAP, which comprises transmembrane segments 2-6. The sterol-dependent interaction between SCAP and Insig is abolished by point mutations in the membranous portion of SCAP (1Yang 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 (768) Google Scholar, 2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar, 7Adams C.M. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10647-10652Crossref PubMed Scopus (89) Google Scholar). These same mutations render cholesterol synthesis resistant to feedback suppression by sterols (8Goldstein J.L. Rawson R.B. Brown M.S. Arch. Biochem. Biophys. 2002; 397: 139-148Crossref PubMed Scopus (193) Google Scholar).Insig proteins also regulate the ER enzyme HMG CoA reductase, which produces mevalonate, the precursor of cholesterol and nonsterol isoprenoids (9Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4500) Google Scholar). Overexpression of the sterol-sensing domain of SCAP prevents the sterol-accelerated, Insig-mediated degradation of HMG CoA reductase (4Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), suggesting that the two proteins bind to the same site on Insigs. The sterol-stimulated binding of HMG CoA reductase to Insig is required for sterol-dependent degradation of the enzyme, as evidenced by the failure of this regulatory process in cells that have been treated with RNAi that target Insig-1 plus Insig-2 (3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar).Human Insig-1 is comprised of 277 amino acids (1Yang 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 (768) Google Scholar), while human Insig-2 contains 225 amino acids (2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar). They demonstrate an amino acid identity of 59%, the differences confined mostly to the hydrophilic N- and C-terminal regions. Insig-2 lacks the N-terminal 50 amino acids of Insig-1. These structural differences are highly conserved across species. Insig-1 and Insig-2 are functionally similar in that both can cause the ER retention of the SCAP/SREBP complex (1Yang 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 (768) Google Scholar, 2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar), and both activate sterol-dependent HMG CoA reductase degradation (3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 4Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Insig-1 expression is positively controlled by nuclear SREBPs, while Insig-2 expression is negatively regulated by insulin (10Yabe D. Komuro R. Liang G. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3155-3160Crossref PubMed Scopus (246) Google Scholar).In order to fully understand the diverse and complex role that Insig proteins play in the regulation of lipid and cholesterol homeostasis, more complete information on the structure and functions of Insigs must be obtained. Sequence inspection suggests that Insig proteins are polytopic membrane-spanning proteins with a high proportion of hydrophobic residues. They are believed to interact with SCAP and HMG CoA reductase through binding to the polytopic membrane-spanning segments of these proteins. Transmembrane segments 2-6 of HMG CoA reductase show clear sequence identities to the sterol-sensing domain of SCAP (3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 11Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) Determining the membrane topology of Insig will provide a map to better explore the mechanism of sterol-induced binding of SCAP and HMG CoA reductase to Insigs.In the current studies we propose and test a model for the topology of Insig-1 through experiments involving protease protection, examination of N-linked glycosylation patterns, and cysteine derivitization. The data are consistent with a model in which Insig-1 contains six membrane-spanning helices separated by very short hydrophilic loops and flanked by short hydrophilic N- and C-terminal extensions that protrude into the cytosol.EXPERIMENTAL PROCEDURESMaterials—We obtained (2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar-(trimethylammonium)ethyl) methanethiosulfonate bromide (MTSET) from Toronto Research Chemicals, Inc.; Nα-(3Sever N. Song B.-L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar-maleimidylpropionyl)biocytin (biotin maleimide) from Molecular Probes, Inc.; Immobilized Neutravidin Biotin Binding Protein from Pierce; and anti-FLAG M2 antibody from Sigma. Monoclonal antibodies IgG-9D5 against hamster SCAP (12Sakai 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 (198) Google Scholar) and IgG-9E10 against c-Myc epitope (1Yang 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 (768) Google Scholar) have been previously described. All other reagents were obtained from previously reported sources (13Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 14Nohturfft 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, 15Rawson R.B. DeBose-Boyd R.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Yabe D. Xia Z.-P. Adams C.M. Rawson R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16672-16677Crossref PubMed Scopus (77) Google Scholar).Plasmids—The following recombinant expression plasmids have been described: pTK-HSV-BP-2, encoding wild-type HSV-tagged human SREBP-2 under control of the thymidine kinase (TK) promoter (17Hua 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); pCMV-SCAP, encoding wild-type hamster SCAP under control of the cytomegalovirus (CMV) promoter (12Sakai 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 (198) Google Scholar); pCMV-Insig-1-MycX6, encoding wild-type human Insig-1 followed by six tandem copies of a c-Myc epitope tag (EQKLISEEDL) under control of the CMV promoter (2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar).pCMV-MycX3-Insig-1 encodes a fusion protein consisting of an initiator methionine, three tandem copies of the c-Myc epitope tag, and amino acids 2-277 of human Insig-1 under control of the CMV promoter. To generate this plasmid, a fragment encoding a BspDI restriction site, amino acids 2-277 of human Insig-1, a stop codon (TGA), and a NotI restriction site was amplified by PCR using pCMV-Insig-1-MycX6 (2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar) as a template, and digested with BspDI and NotI. Forward and reverse primers used for PCR were as follows: 5′-ATGGTATCGATCCCAGATTGCACGACCACTTCTGG-3′ and 5′-TTGCTCAGAGCGGCCGCCTCAATCACTATGGGGCTTTTCAGGAAC-3′ (underlines denote BspDI and NotI sites, respectively). The resulting BspDI-NotI fragment was introduced into pcDNA3 vector between HindIII and NotI sites with a fragment encoding a HindIII restriction site, three tandem copies of the c-Myc epitope tag, and a BspDI restriction site.pCMV-Insig-1-MycX3 encodes a fusion protein consisting of amino acids 1-277 of human Insig-1, followed by three copies of the c-Myc epitope tag under control of the CMV promoter. To generate this plasmid, a BamHI-NotI fragment containing the coding region of human Insig-1 (amino acids 1-277) was released from pCMV-Insig-1-MycX6 (2Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (416) Google Scholar). The resulting BamHI-NotI fragment was introduced into pcDNA3 vector between the BamHI and NotI sites. Then, a fragment encoding a NotI site, three tandem copies of the c-Myc epitope tag, a stop codon (TGA), and a XbaI site were introduced between the NotI and XbaI sites of the newly created plasmid.FLAG versions A-D of pCMV-Insig-1-MycX3 encode proteins similar to wild type, except that two tandem copies of a FLAG epitope tag (DYKDDDDK), flanking a potential glycosylation site (NGT) were introduced between the following two amino acids of human Insig-1: FLAG A, 112 and 113; FLAG B, 61 and 62; FLAG C, 151 and 152; FLAG D, 237 and 238. To generate these plasmids, AgeI sites were first introduced into the corresponding positions of the human Insig-1 coding region by in vitro site-directed mutagenesis using QuickChange XL site-directed mutagenesis kit (Stratagene) with pCMV-Insig-1-MycX3 as a template and the following primers: FLAG A, 5′-CAGAGGAATGTCACCGGTCTCTTCCCCGAG-3′ and 5′-CTCGGGGAAGAGACCGGTGACATTCCTCTG-3′; FLAG B, 5′-GACCCCGCGCCCAGGACCGGTCGCAGTGCTGCG-3′ and 5′-CGCAGCACTGCGACCGGTCCTGGGCGCGGGGTC-3′; FLAG C, 5′-GACAGTCACCTCACCGGTGAACCCCACAAA-3′ and 5′-TTTGTGGGGTTCACCGGTGAGGTGACTGTC-3′; FLAG D, 5′-GTCTATCAGTATACCGGTTCCCCAGATTTC-3′ and 5′-GAAATCTGGGGAACCGGTATACTGATAGAC-3′ (underlines denote AgeI sites).Mutagenesis was confirmed by sequencing. The BamHI-NotI fragment was released from the mutated plasmids and used to replace the corresponding fragment in pCMV-Insig-1-MycX3. The resulting plasmids were digested with AgeI, and a fragment encoding two copies of FLAG epitope tag flanking a potential glycosylation site was introduced into AgeI sites of these plasmids.pCMV-Insig-1-Myc, encoding wild-type human Insig-1 followed by six tandem copies of a c-Myc epitope tag under control of the CMV promoter was used as the template for the generation of all constructs used in cysteine derivitization studies. Briefly, site-directed mutagenesis (Stratagene QuikChange XL), performed according to the manufacturer's suggestions, was used to change all endogenous cysteine residues in Insig-1 to serine (amino acid positions 11, 13, 132, 133, 146, 167, 250). Next, the resultant cysteine-null pCMV-Insig-1-Myc plasmid was subjected to site-directed mutagenesis to introduce individual cysteines at single amino acid positions (S74C, S88C, S93C, V111C, S124C, A184C, T203C, S207C, G212C, V233C, S254C, A267C). All plasmids were sequenced in their entirety prior to use in transfection experiments.Cell Culture—Cells were grown in monolayer at 37 °C in an atmosphere of 8-9% CO2. Human embryonic kidney (HEK)-293 cells were maintained in medium A (Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate). SRD-13A cells are previously described cholesterol and unsaturated fatty acid auxotrophs derived from γ-irradiated Chinese hamster ovary (CHO) cells (15Rawson R.B. DeBose-Boyd R.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and maintained in medium B (a 1:1 mixture of Ham's F-12 medium and DMEM containing 5% fetal calf serum, 5 μg/ml cholesterol, 1 mm sodium mevalonate, 20 μm sodium oleate, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate).Transient Transfection of HEK-293 and SRD-13A Cells—HEK-293 cells were set up on day 0 in medium A at 7 × 105 cells per 100-mm dish. On day 2, the cells were transfected with 4 μg of DNA per dish using a ratio of 12 μl of FuGENE 6 reagent (Roche Applied Science) to 4 μg of DNA in DMEM (without antibiotics) in a final volume of 0.4 ml as follows: FuGENE 6 was diluted in DMEM (without antibiotics), incubated for 5 min at room temperature, and mixed with DNA. This mixture was then further incubated for 15 min at room temperature. Cells were refed with 8 ml of fresh medium A, treated with 0.4 ml of the FuGENE 6/DNA mixture, and incubated at 37 °C for 16-24 h. On day 3, the cells were harvested for cell fractionation.SRD-13A cells were set up on day 0 in medium B at 7 × 105 cells per 60-mm dish. On day 1, the cells were transfected with 4 μg of DNA per dish by using FuGENE 6 reagent as described (15Rawson R.B. DeBose-Boyd R.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). After transfection, cells were incubated at 37 °C for 12 h. On day 3, the cells were washed once with phosphate-buffered saline (PBS), switched to medium C (a 1:1 mixture of Ham's F-12 medium and DMEM containing 5% newborn calf lipoprotein-deficient serum, 50 μm sodium compactin, and 50 μm sodium mevalonate) containing 1% (w/v) hydroxypropyl-β-cyclodextrin. After incubation at 37 °C for 1 h, cells were washed twice with PBS and switched to medium C in the absence or presence of sterols (10 μg/ml cholesterol plus 1 μg/ml 25-hydroxycholesterol added in ethanol (final concentration, 0.5% (v/v)). After incubation for 5 h, cells were harvested for cell fractionation. The total amount of DNA in each transfection was adjusted to 4 μg per dish by addition of pTK mock vector and/or pcDNA3 mock vector.Cell Fractionation—SRD-13A cells were fractionated as described (18DeBose-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 (249) Google Scholar) with minor modifications: The cell pellets from duplicate dishes were resuspended in 0.4 ml of buffer A (10 mm Hepes-KOH (pH 7.4), 10 mm KCl, 1.5 mm MgCl2, 5 mm sodium EDTA, 5 mm sodium EGTA, and 250 mm sucrose), passed through a 22.5-gauge needle 20 times, and centrifuged at 1000 × g for 5 min at 4 °C. The 1000 × g pellets were resuspended in 0.1 ml of buffer B (20 mm Hepes-KOH (pH 7.6), 25% (v/v) glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 5 mm sodium EDTA, 5 mm sodium EGTA), rotated at 4 °C for 1 h, and centrifuged at 105 × g for 30 min at 4 °C in a Beckman TLA 100.2 rotor. The supernatant from this centrifugation was designated the nuclear extract. The supernatant from the original 1000 × g spin was used to prepare the membrane fraction by centrifugation at 105 × g for 30 min at 4 °C. The resulting membrane pellets were resuspended in buffer D (10 mm Tris-HC1 (pH 7.4), 100 mm NaC1, 1% (w/v) SDS).HEK-293 cells were fractionated by the same procedure as SRD-13A cells. To determine the subcellular distribution of Insig-1, the supernatant from the original 1000 × g spin was subjected to centrifugation at 105 × g for 30 min at 4 °C. The supernatant and pellet of this centrifugation was designated the 105 × g supernatant and 105 × g pellet, respectively. To prepare membranes for differential solubilization, protease treatment, glycosidase treatment, and cysteine derivitization, the supernatant from the original 1000 × g spin was subjected to centrifugation at 2 × 104 × g for 15 min at 4 °C. The resulting pellets were resuspended in buffer C (buffer A containing 100 mm NaCl) and designated as the membrane fraction. All the buffers used in the cell fractionation contained protease inhibitors (10 μg/ml leupeptin, 5 μg/ml pepstatin A, 2 μg/ml aprotinin, 25 μg/ml N-acetyl-leucinal-leucinal-norleucinal) except when the membrane fraction was prepared for protease treatment. Protein concentration of the nuclear extracts and membrane fractions was determined using the BCA Kit (Pierce) according to the manufacturer's instructions.Protease Treatment—Trypsin treatment of the membrane fraction was carried out as described (19Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Briefly, aliquots of membranes (75-100 μg of protein) were treated with the indicated amount of trypsin in the absence or presence of 1% (v/v) Triton X-100 in a total volume of 58 μl of buffer C for 30 min at 30 °C. The reactions were then stopped by addition of 2 μl of soybean trypsin inhibitor (400 units), mixed with 5× SDS loading buffer (150 mm Tris-HCl (pH 6.8), 15% SDS, 25% glycerol, 0.02% (w/v) bromphenol blue, 12.5% (v/v) 2-mercaptoethanol), and boiled for 5 min. Proteinase K treatment of the membrane fraction was performed as follows: Aliquots of membranes (100 μg of protein) were treated with the indicated final concentration of proteinase K in the absence or presence of 1% Triton X-100 in a total volume of 58 μl of buffer C for 40 min at 30 °C. The reactions were stopped by addition of 2 μl of phenylmethylsulfonyl fluoride at a final concentration of 5 mm. The samples were then mixed with 5× SDS loading buffer and boiled for 5 min. All the samples were subjected to SDS/PAGE and immunoblot analysis.Glycosidase Treatment—Glycosidase treatment of the membrane fraction was carried out as described (14Nohturfft 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). For treatment with endoglycosidase (endo H) and peptide N-glycosidase F (PNGase F), aliquots of the membranes (60 μg of protein) in 60 μl of buffer C received 10 μl of solution containing 3.5% SDS and 7% 2-mercaptoethanol. After boiling for 10 min, each sample received sequential additions as follows: endo H treatment; 9 μl of 0.5 m sodium citrate (pH 5.5), 5 μl of water, followed by 1 μl of endo H (0.05 units); and PNGase F treatment; 7 μl of 0.5 m sodium phosphate (pH 7.5), 7 μl of 10% (v/v) Nonidet P-40 in water, followed by 1 μl of PNGase F (7.7 × 10-3 units). All reactions were carried out overnight at 37 °C and stopped by addition of 20 μl of 5× SDS loading buffer. The mixtures were then boiled for 5 min and subjected to SDS/PAGE and immunoblot analysis.Cysteine Derivitization—Aliquots of HEK-293 cell membrane fraction (100 μg of protein) were resuspended in 95 μl of buffer C with or without 5 mm (2-(trimethylammonium)ethyl) methanethiosulfonate bromide (MTSET). (A 100 mm stock solution of MTSET was prepared fresh in water.) After incubation for 30 min at room temperature, the membranes were spun twice at 2 × 104 × g for 15 min at 4 °C, washed in 200 μl of buffer C, resuspended in 90 μl of buffer C containing 100 μm biotin maleimide, and incubated for 20 min at room temperature. (A 24 mm stock solution of biotin maleimide was prepared in dimethyl formamide and stored at -20 °C.) At the end of the incubation, the reaction was quenched with 10 mm β-mercaptoethanol (final concentration), the cell membranes were collected by centrifugation, and the pellet was resuspended in 500 μl of buffer E (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1.5% (w/v) Nonidet P-40, 0.1% (w/v) SDS) containing protease inhibitors as described above. Each sample then received a 35-μl aliquot of Immobilized Neutravidin Biotin Binding Protein (washed once in buffer E), after which the samples were incubated overnight under rotation at 4 °C and then centrifuged for 3 min at 200 × g to obtain a supernatant fraction for immunoblot analysis (see below). After removal of the supernatant, the pelleted beads were washed four times in buffer E, resuspended in 100 μl of SDS-containing buffer D, boiled for 5 min, and centrifuged for 30 s at 200 × g to separate the eluted pellet fraction from the beads. Both the supernatant and the eluted pellet fractions were then subjected to SDS/PAGE and immunoblot analysis.Immunoblot Analysis—Samples were subjected to SDS/PAGE, transferred to nitrocellulose filters, and subjected to immunoblot analysis with various antibodies. Gels were calibrated with prestained broad range protein markers (New England BioLabs), and antibodies were used at the following concentrations: anti-Myc IgG-9E10, 1 μg/ml; anti-HSV tag antibody, 0.67 μg/ml; anti-SCAP IgG-9D5, 5 μg/ml; anti-KDEL antibody (StressGen Biotechnologies), 3 μg/ml; anti-transferrin receptor (Zymed Laboratories Inc.), 0.5 μg/ml; anti-FLAG M2 antibody, 1 μg/ml; and anti-mouse IgG (Jackson ImmunoResearch), 0.2 μl/ml. Bound antibodies were visualized by chemiluminescence using the Supersignal Substrate System (Pierce) according to manufacturer's instructions. Filters were exposed to Kodak X-Omat Blue XB-1 films at room temperature for various times as indicated in legends.RESULTSFig, 1A shows a hydropathy plot of the amino acid sequence of human Insig-1. Based on this plot, Insig-1 is predicted to contain approximately six transmembrane segments, depicted by peak regions of hydrophobicity. Based on data from experiments described below, we suggest a membrane topology for human Insig-1 that is shown in Fig. 1B. The membrane-spanning segments correspond to hydrophobic sequences 1-6 in the hydropathy plot. The hydrophilic N-terminal and C-terminal ends are proposed to extend into the cytosol with the remaining portion, containing six membrane-spanning helices, embedded in the membrane.As a first step in these studies, we documented the membrane association of Insig-1 by transfecting human embryonic kidney-293 (HEK-293) cells with pCMV-Insig-1-MycX6, which encodes human Insig-1 with six copies of a c-Myc epitope tag at its C terminus (Fig. 2). Previous studies demonstrated that this epitope-tagged version of Insig-1 has wild-type activity in its ability to bind SCAP in a sterol-dependent fashion and" @default.
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• W2005218056 date "2004-02-01" @default.
• W2005218056 modified "2023-10-16" @default.
• W2005218056 title "Membrane Topology of Human Insig-1, a Protein Regulator of Lipid Synthesis" @default.
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