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• W2040042360 abstract "Although a critical role of microsomal transfer protein (MTP) has been recognized in the assembly of nascent apolipoprotein B (apoB)-containing lipoproteins, it remains unclear where and how MTP transfers lipids in the secretory pathway during the maturational process of apoB lipidation. The aims of this study were to determine whether MTP functions in the secretory pathway as well as in the endoplasmic reticulum and whether its large 97-kDa subunit interacts with the small 58-kDa protein disulfide isomerase (PDI) subunit and apoB, particularly in the Golgi apparatus. Using a high resolution immunogold approach combined with specific polyclonal antibodies, the large and small subunits of MTP were observed over the rough endoplasmic reticulum and the Golgi. Double immunocytochemical detection unraveled the colocalization of MTP and PDI as well as MTP and apoB in these same subcellular compartments. To confirm the spatial contact of these proteins, Golgi fractions were isolated, homogenized, and incubated with an anti-MTP large subunit antibody. Immunoprecipitates were applied on SDS-PAGE and then transferred on to nitrocellulose. Immunoblotting the membrane with PDI and apoB antibodies confirmed the colocalization of these proteins with MTP. Furthermore, MTP activity assay disclosed a substantial triglyceride transfer in the Golgi fractions. The occurrence of membrane-associated apoB in the Golgi, coupled with its interaction with active MTP, suggests an important role for the Golgi in the biogenesis of apoB-containing lipoproteins. Although a critical role of microsomal transfer protein (MTP) has been recognized in the assembly of nascent apolipoprotein B (apoB)-containing lipoproteins, it remains unclear where and how MTP transfers lipids in the secretory pathway during the maturational process of apoB lipidation. The aims of this study were to determine whether MTP functions in the secretory pathway as well as in the endoplasmic reticulum and whether its large 97-kDa subunit interacts with the small 58-kDa protein disulfide isomerase (PDI) subunit and apoB, particularly in the Golgi apparatus. Using a high resolution immunogold approach combined with specific polyclonal antibodies, the large and small subunits of MTP were observed over the rough endoplasmic reticulum and the Golgi. Double immunocytochemical detection unraveled the colocalization of MTP and PDI as well as MTP and apoB in these same subcellular compartments. To confirm the spatial contact of these proteins, Golgi fractions were isolated, homogenized, and incubated with an anti-MTP large subunit antibody. Immunoprecipitates were applied on SDS-PAGE and then transferred on to nitrocellulose. Immunoblotting the membrane with PDI and apoB antibodies confirmed the colocalization of these proteins with MTP. Furthermore, MTP activity assay disclosed a substantial triglyceride transfer in the Golgi fractions. The occurrence of membrane-associated apoB in the Golgi, coupled with its interaction with active MTP, suggests an important role for the Golgi in the biogenesis of apoB-containing lipoproteins. Lipids constitute the most calorically dense dietary nutrients. They must undergo emulsification within the intestinal lumen, cell membrane permeation, intracellular esterification, and incorporation into chylomicrons before reaching the circulatory system (for reviews, see Refs. 1.Davidson N.O. Johnson L.R. Physiology of Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1909-1934Google Scholar, 2.Tso P. Johnson L.R. Physiology of Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1867-1907Google Scholar, 3.Levy E. Can. J. Physiol. Pharmacol. 1992; 70: 413-419Crossref PubMed Scopus (33) Google Scholar, 4.Hussain M.M. Kancha R.K. Zhou Z. Luchoomun J. Zu H. Bakillah A. Biochim. Biophys. Acta. 1996; 1300: 151-170Crossref PubMed Scopus (146) Google Scholar). Despite significant progress, our understanding of the complex biosynthetic process involved in the formation and secretion of triglyceride-rich lipoprotein particles remains rather fragmentary. In particular, we know little about the sequential multistep assembly of apolipoproteins and lipids or the topology of the proteins in intracellular organelles implicated in lipoprotein production. The study of naturally occurring mutations and genetic variations in humans has greatly contributed to the identification of the proteins essential to the synthetic pathway and to the delineation of key metabolic mechanisms (5.Levy E. Clin. Invest. Med. 1996; 19: 317-324PubMed Google Scholar, 6.Levy E. Marcel R. Milne R.W. Grey V.L. Roy C. Gastroenterology. 1987; 93: 1119-1126Abstract Full Text PDF PubMed Google Scholar, 7.Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (639) Google Scholar, 8.Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M. Jarmuz A. Grantham T.T. Leoni P.R. Bhattacharya S. Pease R.J. Cullen P.M. Levi S. Byfield P.G.H. Purkiss P. Scott J. Hum. Mol. Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (227) Google Scholar, 9.Black D.D. Hay R.V. Rohwer-Nutter P.L. Ellinas H. Stephens J.K. Sherman H. Teng B.B. Whitington P.F. Davidson N.O. Gastroenterology. 1991; 101: 520-528Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 10.Young S.G. Krul E.S. McCormick S. Farese Jr., R.V. Linton M.F. Methods Enzymol. 1996; 263: 120-145Crossref PubMed Scopus (7) Google Scholar, 11.Wu J. Kim J. Li Q. Kwok P.-Y. Cole T.G. Cefalu B. Averna M. Schonfeld G. J. Lipid Res. 1999; 40: 955-959Abstract Full Text Full Text PDF PubMed Google Scholar). Inherited disorders of apolipoprotein B (apoB) 1The abbreviations used are: apoBapolipoprotein BMTPmicrosomal transfer proteinERendoplasmic reticulumVLDLvery low density lipoproteinPDIprotein disulfide isomerasePBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and microsomal transfer protein (MTP) deficiency have provided a unique source for delineating, at least partially, the role of these specific proteins as well as elucidating the intracellular mechanisms that result in lipid absorption and transport (5.Levy E. Clin. Invest. Med. 1996; 19: 317-324PubMed Google Scholar, 6.Levy E. Marcel R. Milne R.W. Grey V.L. Roy C. Gastroenterology. 1987; 93: 1119-1126Abstract Full Text PDF PubMed Google Scholar, 7.Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (639) Google Scholar, 8.Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M. Jarmuz A. Grantham T.T. Leoni P.R. Bhattacharya S. Pease R.J. Cullen P.M. Levi S. Byfield P.G.H. Purkiss P. Scott J. Hum. Mol. Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (227) Google Scholar, 9.Black D.D. Hay R.V. Rohwer-Nutter P.L. Ellinas H. Stephens J.K. Sherman H. Teng B.B. Whitington P.F. Davidson N.O. Gastroenterology. 1991; 101: 520-528Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 10.Young S.G. Krul E.S. McCormick S. Farese Jr., R.V. Linton M.F. Methods Enzymol. 1996; 263: 120-145Crossref PubMed Scopus (7) Google Scholar, 11.Wu J. Kim J. Li Q. Kwok P.-Y. Cole T.G. Cefalu B. Averna M. Schonfeld G. J. Lipid Res. 1999; 40: 955-959Abstract Full Text Full Text PDF PubMed Google Scholar). The addition of core lipid to the nascent lipoprotein particle is thought to occur in conjunction with the translation and translocation of apoB in the ER (12.Boren J. Graham L. Wettesten M. Scott J. White A. Olofsson S.-O. J. Biol. Chem. 1992; 267: 9858-9867Abstract Full Text PDF PubMed Google Scholar, 13.Rustaeus S. Lindberg K. Boren J. Olofsson S.-O. J. Biol. Chem. 1995; 270: 28879-28886Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 14.Spring D.J. Chem-Liu L.W. Chatterton J.E. Elovson J. Schumaker V.N. J. Biol. Chem. 1992; 267: 14839-14845Abstract Full Text PDF PubMed Google Scholar, 15.McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar). During this process, apoB remains tightly bound to the ER membrane, where it is folded. The initial complement of lipid is then added to form a nascent, small, dense lipoprotein particle. In the second step, maturation of the particle occurs by the addition of the neutral lipid core (16.Swift L.L. J. Lipid Res. 1995; 36: 395-406Abstract Full Text PDF PubMed Google Scholar, 17.Boren J. Rustaeus S. Olofsson S.-O. J. Biol. Chem. 1994; 269: 25879-25888Abstract Full Text PDF PubMed Google Scholar). Based upon the known lipid transfer activity of MTP, its localization in the lumen of the ER, and the observation that apoB is degraded intracellularly and not secreted in the absence of MTP, it has been proposed that MTP shuttles lipids from the ER membrane to the growing apoB chain in the ER, allowing the protein to translocate completely into the lumen (18.Gordon D.A. Jamil H. Gregg R.E. Olofsson S.-O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 19.Wang Y. McLeod R.S. Yao Z. J. Biol. Chem. 1997; 262: 12272-12278Abstract Full Text Full Text PDF Scopus (86) Google Scholar, 20.Benoist F. Grand-Perret T. J. Biol. Chem. 1997; 272: 20435-20442Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, very little is known about where and how the addition of bulk lipids to the nascent particle takes place. apolipoprotein B microsomal transfer protein endoplasmic reticulum very low density lipoprotein protein disulfide isomerase phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Several models have been proposed for the formation of VLDL: (a) complete assembly of VLDL in the ER (21.Alexander C.A. Hamilton R.L. Havel R.J. J. Cell Biol. 1976; 69: 241-263Crossref PubMed Scopus (253) Google Scholar, 22.Borchardt R.A. Davis R.A. Biol. Chem. 1987; 262: 16394-16402Abstract Full Text PDF Google Scholar, 23.Rusinol A. Verkade H. Vance J.E. J. Biol. Chem. 1993; 268: 3555-3562Abstract Full Text PDF PubMed Google Scholar); (b) association of apoB with membranes until it reaches the Golgi apparatus, whereupon lipid is added to the particle (24.Bamberger M.J. Lane M.D. J. Biol. Chem. 1988; 263: 11868-11878Abstract Full Text PDF PubMed Google Scholar, 25.Bamberger M.J. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2390-2394Crossref PubMed Scopus (47) Google Scholar); and (c) a sequential addition of lipid to apoB during its passage from the ER to the Golgi for secretion (26.Bostrom K. Boren J. Wettesten M. Sjoberg A. Bondjers G. Wiklund O. Carlsson P. Olofsson S.O. J. Biol. Chem. 1988; 263: 4434-4442Abstract Full Text PDF PubMed Google Scholar, 27.Bostrom K. Wettesten M. Boren J. Bondjers G. Wiklund O. Olofsson S.O. J. Biol. Chem. 1986; 261: 13800-13806Abstract Full Text PDF PubMed Google Scholar, 28.Janero D.R. Lane M.D. J. Biol. Chem. 1983; 258: 14496-14504Abstract Full Text PDF PubMed Google Scholar). If partial assembly takes place in the Golgi and additional core lipids and phospholipids are added in the pre-Golgi and Golgi as has been suggested (25.Bamberger M.J. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2390-2394Crossref PubMed Scopus (47) Google Scholar, 29.Hamilton R.L. Moorehouse A. Havel R.J. J. Lipid Res. 1991; 32: 529-543Abstract Full Text PDF PubMed Google Scholar), one would anticipate the obligatory presence of MTP in these compartments. Structurally, MTP is a heterodimer composed of a unique large subunit (97 kDa) and a smaller subunit (58 kDa) (30.Wetterau J.R. Zilversmit D.B. Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (100) Google Scholar, 31.Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar, 32.Wetterau J.R. Aggerbeck L.P. Laplaud P.M. McLean L.R. Biochemistry. 1991; 30: 4406-4412Crossref PubMed Scopus (77) Google Scholar, 33.Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (155) Google Scholar). The latter has been identified as the multifunctional enzyme, protein disulfide isomerase (PDI). The role of PDI in the function of MTP is not entirely clear. The two subunits form a tight complex, and their dissociation eliminates MTP activity. The intracellular location of MTP in the intestine has not been thoroughly elucidated. Subfractionation of crude liver homogenates suggested that MTP is located in hepatocellular microsomes (32.Wetterau J.R. Aggerbeck L.P. Laplaud P.M. McLean L.R. Biochemistry. 1991; 30: 4406-4412Crossref PubMed Scopus (77) Google Scholar, 33.Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (155) Google Scholar). Nevertheless, this finding was not confirmed by immunoelectron microscopy. In the present paper, the following issues are addressed. 1) Can MTP be found in secretory pathway sites other than the ER? 2) Does it occur alone or in close proximity to PDI in these compartments? 3) Does it colocalize with apoB? 4) If MTP can be detected in the Golgi apparatus, is it functional? 5) Can it facilitate apoB translocation from Golgi membranes? The answers to these questions provided by the experiments described herein help elucidate the mechanisms involved in the assembly and secretion of apoB-containing lipoproteins. Sprague-Dawley rats were used for all experiments. Jejunal specimens were taken at the ligament of Treitz, washed, and prepared for microscopy visualization. For the isolation of Golgi, the mucosa was scraped, homogenized, and ultracentrifuged. Intestinal specimens were fixed by immersion in 1% glutaraldehyde, 0.1m phosphate-buffered saline (pH 7.4) for 2 h at 4 °C and embedded in Lowicryl K4M at −20 °C according to our previously described procedures (34.Bendayan M. J. Electron Microsc. Technol. 1984; 1: 243-270Crossref Scopus (370) Google Scholar). Tissue blocks were examined by light microscopy to select well oriented villus tips. Thin sections (60–80 nm) of the different tissue blocks were mounted on nickel grids with a carbon-coated Parlodion film and processed for immunocytochemistry. Protein A-gold immunocytochemical techniques were employed to detect the presence of MTP, PDI, and apoB in rat intestinal tissue as we have described previously (34.Bendayan M. J. Electron Microsc. Technol. 1984; 1: 243-270Crossref Scopus (370) Google Scholar, 35.Levy E. Rochette C. Londono I. Roy C.C. Milne R.W. Marcel Y.L. Bendayan M. J. Lipid Res. 1990; 31: 1937-1946Abstract Full Text PDF PubMed Google Scholar). Briefly, the tissue sections were washed initially in distilled water, incubated for 5 min on a drop of PBS containing 1% ovalbumin, and transferred subsequently to a drop of the PBS-diluted antibody (see below). After incubation (90 min) at room temperature, the grids were rinsed with PBS to remove unbound antibodies. They were transferred to the PBS-ovalbumin (3 min) and incubated on a drop of protein A-gold (pH 7.2) for 30 min at room temperature. The tissue sections were then thoroughly washed with PBS, rinsed with distilled water, and dried. Sections were stained with uranyl acetate and lead citrate before examination with a Philips 410 electron microscope. Polyclonal antibodies were used at various dilutions (MTP 1/100, MTP large subunit 1/50, PDI 1/10, apoB 1/100) in combination with protein A-gold complexes, which were prepared using 10- or 5-nm gold particles according to our established techniques (35.Levy E. Rochette C. Londono I. Roy C.C. Milne R.W. Marcel Y.L. Bendayan M. J. Lipid Res. 1990; 31: 1937-1946Abstract Full Text PDF PubMed Google Scholar, 36.Bendayan M. Prog. Histochem. Cytochem. 1995; 29: 1-163Crossref PubMed Scopus (158) Google Scholar). Control experiments were performed to assess the specificity of the results. Excess purified MTP (10-fold) was added to the antibody solution. Incubation with this solution was followed by the protein A-gold complex. Pre-immune rabbit serum (diluted 1:10) was used on tissue sections before incubation with protein A-gold complex. Incubations were also performed with the protein A-gold complex alone, omitting the antibody step to test for nonspecific adsorption of the protein A-gold complex to tissue sections (36.Bendayan M. Prog. Histochem. Cytochem. 1995; 29: 1-163Crossref PubMed Scopus (158) Google Scholar). To reveal the existence of MTP-PDI as well as MTP-ApoB complexes within the cellular compartments, the double-labeling technique was applied. The tissue sections were labeled simultaneously for either MTP and PDI or MTP and ApoB. The two-phase labeling technique (36.Bendayan M. Prog. Histochem. Cytochem. 1995; 29: 1-163Crossref PubMed Scopus (158) Google Scholar, 37.Bendayan M. J. Histochem. Cytochem. 1982; 30: 81-85Crossref PubMed Scopus (291) Google Scholar) was applied to avoid any cross-reaction between reagents. The small protein A-gold complex (5 nm) was used for the first labeling protocol, and the larger 10 nm protein A-gold complex was used for the second. This protocol allows for the simultaneous visualization of two antigens (MTP and PDI or MTP and ApoB) in the same tissue section. The antibodies for the MTP, PDI, and MTP large subunits were kindly provided by John Wetterau, Harris Jamil, and one of the authors (C. Shoulders). These antibodies have been characterized and utilized successfully in previous studies (7.Wetterau J.R. Aggerbeck L.P. Bouma M.E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (639) Google Scholar, 31.Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar, 38.Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R. Wetterau J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7628-7632Crossref PubMed Scopus (189) Google Scholar,39.Leiper J.M. Mayliss J.D. Perse R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar). The antibody directed against rat apoB was raised in rabbits (35.Levy E. Rochette C. Londono I. Roy C.C. Milne R.W. Marcel Y.L. Bendayan M. J. Lipid Res. 1990; 31: 1937-1946Abstract Full Text PDF PubMed Google Scholar). Confirmation of the specificity of the antibodies was obtained when: 1) MTP large and small subunits as well as apoB from total homogenate were fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with each polyclonal antibody; and 2) cellular lysates were first reacted with the antibodies (directed against large MTP subunit, small MTP subunit, and apoB antibodies) before the immunoprecipitates were subjected to gel electrophoresis, transferred to nitrocellulose membranes, and reacted with the underlying antibodies. Microsomal and Golgi fractions were prepared from enterocytes of fasted rats using modifications of reported techniques (40.Levy E. Garofalo C. Rouleau T. Gavino V. Bendayan M. Hepatology. 1996; 23: 848-857Crossref PubMed Google Scholar, 41.Marks D.L. Wu K. Paul P. Kamisaka Y. Watanabe R. Pagano R.E. J. Biol. Chem. 1999; 274: 451-456Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Epithelial cells were homogenized (25%, w/v) in 0.25 m sucrose in buffer (50 mm Tris-HCl, pH 7.4, and 25 mm KCl) containing protease inhibitors (10 μg/ml leupeptin, 10 μg aprotinin, 1 μg/ml pepstatin, 1 μg/ml antipain, and 25 mm4-aminophenyl-methanesulfonyl fluoride) using a polytron (Brinkmann Instruments) at a setting of 1 (30 s). The homogenate from a group of animals was used to prepare microsomes representative of the endoplasmic reticulum (40.Levy E. Garofalo C. Rouleau T. Gavino V. Bendayan M. Hepatology. 1996; 23: 848-857Crossref PubMed Google Scholar). For the preparation of Golgi fractions, the homogenate from another group of animals was filtered through cheesecloth and then adjusted (150 parts homogenate and 95 parts 2m sucrose in the aforementioned buffer) to a final concentration of 1.07 m sucrose. The adjusted homogenate (19 ml/tube) was loaded into Beckman SW 28 tubes; 9 ml each of 0.9 and 0.2 m sucrose in the aforementioned buffer were then sequentially overlaid above the homogenate. The tubes were subsequently centrifuged in an SW 28 rotor for 2 h at 83,000 ×g. Golgi fractions were collected at the 0.2/0.9m sucrose interface. To prepare Golgi membranes, Golgi fractions were mixed 1:1 (v/v) with the buffer (50 mm HEPES, pH 7.4, 100 mm KCl, and the protease inhibitors as indicated above) with 20% glycerol and centrifuged for 90 min at 200,000 × g. Golgi membrane pellets were resuspended at 5% of their original volume in 0.25m sucrose in the same buffer. Golgi membranes were solubilized with either 1% Igepal CA 630 (a Nonidet P-40 equivalent from Sigma) or 1% CHAPS and 0.2% Triton X-100-containing buffer. The extract was then centrifuged at 200,000 × g, and the pellet was discarded. The purity of the Golgi subcellular fraction was verified by assay of galactosyltransferase, a specific marker for Golgi organelles, and glucose-6-phosphatase, a marker for the ER. The intestinal ER and Golgi specimens were sonicated in 1 ml of 10 mm phosphate buffer (pH 6.8) containing saponin (100 μg/ml) and protease inhibitors (leupeptin, 10 μg/ml; Trasylol, 10 μg/ml; pepstatin A, 1 μg/ml). The MTP/PDI heterodimer was separated by ultracentrifugation (100,000 × g) and concentrated using a Centricon 30 cartridge (5000 rpm × 30 min). The triglyceride transfer assay was adapted from previous reports (30.Wetterau J.R. Zilversmit D.B. Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (100) Google Scholar, 39.Leiper J.M. Mayliss J.D. Perse R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar, 42.Narcisi T.M. Shoulders C.C. Chester S.A. Read J. Brett D.J. Harrison G.B. Grantham T.T. Fox M.F. Povey S. de Bruin T.W. Ekerlens D.W. Muller D.P.R. Lloyd J.K. Scott J. Am. J. Hum. Genet. 1995; 57: 1298-1310PubMed Google Scholar). The MTP transfer activity was determined by evaluating the transfer of radiolabeled triacylglycerol between two populations of unilamellar vesicles as described (30.Wetterau J.R. Zilversmit D.B. Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (100) Google Scholar, 39.Leiper J.M. Mayliss J.D. Perse R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar, 43.Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar). The donor and receiver vesicles were prepared by adding the appropriate amount of lipids to 500 μl of chloroform followed by drying under a stream of nitrogen, rehydration, and probe sonication in 1.25 ml of 15:35 buffer (15 mm Tris/HCl, pH 7.4, 35 mm NaCl, 0.05% bovine serum albumin, 3 mm sodium azide, 1 mm EDTA). Donor vesicles contained, per assay, 4 nmol of egg yolk phosphatidylcholine, 0.33 nmol of cardiolipin, and 0.024 nmol of [3H]trioleylglycerol (Amersham Biosciences). Receptor vesicles contained 24 nmol of egg yolk phosphatidylcholine, 0.048 nmol of trioleylglycerol, and ∼4000 cpm of [14C]dipalmitoyl phosphatidylcholine ([14C]DPPC; Amersham Biosciences). Both categories of vesicles comprised 0.01% butylated hydroxytoluene. Various amounts of semi-purified MTP were incubated with 5 μl of donor and receptor vesicles in a final volume of 100 μl for 1 h at 37 °C. The reaction was quenched by adding 400 μl of ice-cold 15:35 buffer (without bovine serum albumin). The negatively charged (due to the presence of cardiolipin) donor vesicles were removed from the reaction mixture by adsorption onto DEAE-cellulose (Whatman DE-52). The supernatant (containing the receptor vesicles) was collected after a low speed centrifugation (13000 × g) and recentrifuged (13000 × g) to assure a total removal of the DEAE-cellulose before scintillation counting. A blank assay containing donor and acceptor membranes without transferred protein was used to correct for the spontaneous transfer of labeling between vesicles. The ratio of [3H]glycerol trioleate on [14C]dipalmitoyl phosphatidylcholine was determined, and the percentage lipid transfer was calculated from the increase in this ratio. Lipid transfer activity was determined from the initial linear portions of the activity curves. ApoB synthesis was assessed in everted rat intestine, as described previously (44.Levy E. Garofalo C. Thibault L. Dionne S. Daoust L. Lepage G. Roy C.C. Am. J. Physiol. 1992; 262: G319-G326PubMed Google Scholar). Then, 300 μCi of [35S]methionine was added to the RPMI 1640 medium for 1 h. The medium was removed, and the intestine was washed and again incubated for 2 h with medium containing 10 mm unlabeled methionine in the presence or absence of 15 μm BMS-200150, an inhibitor of MTP. Labeled apoB from membranes and the luminal contents of isolated microsomal and Golgi fractions were immunoprecipitated, subjected to SDS-PAGE electrophoresis, excised from the gel, and counted (45.Levy E. Sinnett D. Thibault L. Nguyen T.D. Delvin E. Ménard D. FEBS Lett. 1996; 393: 253-258Crossref PubMed Scopus (58) Google Scholar). The first step in our studies was to test the specificity of the polyclonal antibodies that were generously provided by investigators (see “Acknowledgments”) who remarkably advanced the knowledge of MTP. By immunoprecipitating epithelial cell lysates, separating the immunoprecipitates on SDS-PAGE, and Western blotting them separately with large 97-kDa and small 58-kDa antibodies, we identified the expected 97- and 58-kDa subunits corresponding to the MTP and PDI components, respectively (Fig. 1 A). The use of polyclonal antibodies directed against the whole MTP complex resulted in the recovery of both MTP and PDI subunits (Fig. 1 A). Similar experimental procedures displayed and confirmed the specificity of anti-apoB antibodies (Fig. 1 A). In a second step, the specificity of antibodies was verified by fractionating the total homogenate by SDS-PAGE, transferring it to nitrocellulose, and immunoblotting. Once again, we found only the signal corresponding to the targeted protein (Fig. 1 B). We subsequently used immunocytochemical techniques and isolated microsomal and Golgi fractions to reveal the presence of MTP in these subcellular compartments. The purity of microsomal and Golgi fractions was determined by the assay of galactosyltransferase, as a specific marker for Golgi membranes, and glucose-6-phosphatase as a marker for the ER. The results of marker protein assays from a typical fractionation are shown in Table I. The Golgi fraction was enriched 96-fold with UDP-galactose galactosyltransferase specific activity over cell homogenate, whereas glucose-6-phosphatase activity was very low (1.26-fold) in this cell organelle. Furthermore, the rough ER-derived fraction was enriched 3.8-fold in the specific activity of glucose-6-phosphotase and 2.46-fold in that of galactosyltransferase.Table ISpecific activities of marker enzymes of subcellular fractionsOrganelleGalactosyltransferaseGlucose-6-phosphatasenmol/mg protein/minWhole homogenate0.041 (×1.00)0.720 (×1.00)Microsomal fraction0.101 (×2.46)2.735 (×3.80)Golgi fraction3.926 (×95.75)0.907 (×1.26)Microsomal and Golgi subcellular fractions were prepared as described under “Materials and Methods.” The purity of the organelle fractions was assessed by determining the specific activities of UDP-galactose galactosyltransferase and glucose-6-phosphatase. The enrichment of the marker enzymes in each cellular fraction was calculated by dividing the specific activity of the subcellular fraction by that of the homogenate (data are given in parentheses). The data represent the average of n = 2. Open table in a new tab Microsomal and Golgi subcellular fractions were prepared as described under “Materials and Methods.” The purity of the organelle fractions was assessed by determining the specific activities of UDP-galactose galactosyltransferase and glucose-6-phosphatase. The enrichment of the marker enzymes in each cellular fraction was calculated by dividing the specific activity of the subcellular fraction by that of the homogenate (data are given in parentheses). The data represent the average of n = 2. An anti-MTP heterodimer antibody was initially applied to define the immunocytochemical pattern of labeling in enterocytes (Fig. 2). The ultrastructural analysis of rat enterocytes revealed significant labeling over the rough ER, the Golgi area, and basolateral membranes (Fig. 2). The trans-Golgi cisternae were more intensely labeled. Finally, the gold particles in the basal region of the enterocyte were associated with the basolateral membrane and its interdigitations (Fig. 2). Only very few particles were located over the mitochondria and nuclei. Control experiments confirmed the specificity of these results. The preadsorbtion of the antibody with its antigen prior to performing immunocytochemical detection resulted in very low labeling in all cellular regions (Fig. 3). Similar data were obtained with the other control experiments, confirming the validity of the morphological findings. Furthermore, the addition of albumin to the antibody solution did not alter the pattern of labeling. The antibody used in this first series of experiments recognizes the whole 97-kDa-58-kDa MTP protein complex. We therefore attempted to distinguish between the two subunits by employing antibodies specific to the 97-kDa and the 58-kDa polypeptides separately. With the antibody directed against the MTP large subunit, the distribution of the labeling (Fig. 4) over the rough ER and the Golgi" @default.
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• W2040042360 cites W1551374056 @default.
• W2040042360 cites W1554617882 @default.
• W2040042360 cites W1591366077 @default.
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• W2040042360 cites W1657755666 @default.
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• W2040042360 cites W1973856139 @default.
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• W2040042360 cites W1976995910 @default.
• W2040042360 cites W1981660336 @default.
• W2040042360 cites W1985826486 @default.
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• W2040042360 cites W2004023727 @default.
• W2040042360 cites W2009997321 @default.
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• W2040042360 cites W2031426969 @default.
• W2040042360 cites W2042381594 @default.
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• W2040042360 cites W2054407624 @default.
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• W2040042360 cites W2083520071 @default.
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