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A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B

2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês

10.1074/jbc.m300271200

ISSN

1083-351X

Autores

Jeremy A. Sellers, Li Hou, Humra Athar, M. Mahmood Hussain, Gregory S. Shelness,

Tópico(s)

Plant biochemistry and biosynthesis

Resumo

The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of ∼1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism. The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of ∼1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism. Processes responsible for the efficient capture, transport, and storage of lipids are observed in all multicellular organisms. In vertebrates, apoB 1The abbreviations used are: apoB, apolipoprotein B; apoLp-II/I, apolipophorin II/I; AP, alkaline phosphatase; DMEM, Dulbecco's modified Eagle's media; EST, expressed sequence tag; HDLp, high density lipophorin; MTP, microsomal triglyceride transfer protein; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TG, Triglyceride; VLDL, very low density lipoprotein; ELISA, enzyme-linked immunosorbent assay. plays a complex role in lipid utilization, beginning with the enterocytic assembly of dietary lipids to form chylomicron particles (1Hussain M.M. Atherosclerosis. 2000; 148: 1-15Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The liver is the second major site of apoB expression where it assembles endogenous lipids to form triglyceride (TG)-rich very low density lipoproteins (VLDL) (2Davidson N.O. Shelness G.S. Annu. Rev. Nutr. 2000; 20: 169-193Crossref PubMed Scopus (235) Google Scholar). Both chylomicrons and VLDL function to distribute TG to peripheral tissues; however, metabolic products of these particles can accumulate in plasma and contribute to several chronic disease states, including atherosclerosis and diabetes (3Goldstein J.L. Brown M.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. McGraw-Hill, New York1989: 1215-1250Google Scholar, 4Kannel W.B. Marmot M. Elliott P. Coronary Heart Disease Epidemiology. From Aetiology to Public Health. Oxford University Press, New York1992: 67-82Google Scholar). The discovery of MTP as a critical cofactor essential for chylomicron and VLDL formation has provided considerable mechanistic insight into the processes responsible for intracellular lipoprotein assembly (5Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar, 6Gordon D.A. Jamil H. Biochim. Biophys. Acta Mol. Cell. Biol. 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Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar, 11Anderson T.A. Levitt D.G. Banaszak L.J. Structure. 1998; 6: 895-909Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12Segrest J.P. Jones M.K. Dashti N. J. Lipid Res. 1999; 40: 1401-1416Abstract Full Text Full Text PDF PubMed Google Scholar). Vitellogenin is an ancient lipid-binding protein that functions in the transport of lipids and other nutrients (13Ohlendorf D.H. Barbarsh G.R. Trout A Kent C. Banaszak L.J. J. Biol. Chem. 1977; 252: 7992-8001Abstract Full Text PDF PubMed Google Scholar) from liver or fat body to the developing oocyte of oviparous vertebrates and invertebrates (14Wahli W. Trends Genet. 1988; 4: 227-232Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 15Byrne B.M. Gruber M. AB G. Prog. Biophys. Mol. Biol. 1989; 53: 33-69Crossref PubMed Scopus (284) Google Scholar). Another form of primitive lipoprotein biogenesis is the assembly of apolipophorin II/I (apoLp-II/I) into high density lipophorin (HDLp) particles (16Canavoso L.E. Jouni Z.E. Karnas K.J. Pennington J.E. Wells M.A. Annu. Rev. Nutr. 2001; 21: 23-46Crossref PubMed Scopus (464) Google Scholar, 17Weers P.M.M. Van der Horst D.J. Van Marrewijk W.J.A. Van den Eijnden M.V. Van Doorn J.M. Beenakkers A.M.T. J. Lipid Res. 1992; 33: 485-491Abstract Full Text PDF PubMed Google Scholar). Unlike apoB-containing particles, whose initial formation and ongoing enlargement occurs exclusively within the secretory pathway, maturation of HDLp is a post secretory event that is achieved by efflux of diglycerides from insect fat body to the HDLp acceptor. The mature lipophorin, termed low density lipophorin, is then delivered to flight muscle where its lipid is discharged and used as an energy source. Although apoLp-II/I is also a homolog of apoB (18Babin P.J. Bogerd J. Kooiman F.P. Van Marrewijk W.J.A. 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Charlab R. Nusskern D.R. Wincker P. Clark A.G. Ribeiro J.M. Wides R. Salzberg S.L. Loftus B. Yandell M. Majoros W.H. Rusch D.B. Lai Z. Kraft C.L. Abril J.F. Anthouard V. Arensburger P. Atkinson P.W. Baden H. de Berardinis V. Baldwin D. Benes V. Biedler J. Blass C. Bolanos R. Boscus D. Barnstead M. Cai S. Center A. Chatuverdi K. Christophides G.K. Chrystal M.A. Clamp M. Cravchik A. Curwen V. Dana A. Delcher A. Dew I. Evans C.A. Flanigan M. Grundschober-Freimoser A. Friedli L. Gu Z. Guan P. Guigo R. Hillenmeyer M.E. Hladun S.L. Hogan J.R. Hong Y.S. Hoover J. Jaillon O. Ke Z. Kodira C. Kokoza E. Koutsos A. Letunic I. Levitsky A. Liang Y. Lin J.J. Lobo N.F. Lopez J.R. Malek J.A. McIntosh T.C. Meister S. Miller J. Mobarry C. Mongin E. Murphy S.D. O'Brochta D.A. Pfannkoch C. Qi R. Regier M.A. Remington K. Shao H. Sharakhova M.V. Sitter C.D. Shetty J. Smith T.J. Strong R. Sun J. Thomasova D. Ton L.Q. Topalis P. Tu Z. Unger M.F. Walenz B. Wang A. Wang J. Wang M. Wang X. Woodford K.J. Wortman J.R. Wu M. Yao A. Zdobnov E.M. Zhang H. Zhao Q. Zhao S. Zhu S.C. Zhimulev I. Coluzzi M. della Torre A. Roth C.W. Louis C. Kalush F. Mural R.J. Myers E.W. Adams M.D. Smith H.O. Broder S. Gardner M.J. Fraser C.M. Birney E. Bork P. Brey P.T. Venter J.C. Weissenbach J. Kafatos F.C. Collins F.H. Hoffman S.L. Science. 2002; 298: 129-149Crossref PubMed Scopus (1616) Google Scholar). When the Drosophila data base was searched for sequences similar to human MTP a single gene, CG9342, was identified. Given its 23% identity with human MTP, this gene product was initially described as a triglyceride-binding protein (22Stapleton M. Liao G. Brokstein P. Hong L. Carninci P. Shiraki T. Hayashizaki Y. Champe M. Pacleb J. Wan K. Yu C. Carlson J. George R. Celniker S. Rubin G.M. Genome Res. 2002; 12: 1294-1300Crossref PubMed Scopus (120) Google Scholar). In this report we functionally characterized CG9342 and observed that, in transfected cells, it supports the assembly and secretion of human apoB34 and apoB41 as lipoprotein precursors. In addition, we observed functional and structural differences between CG9342 and human MTP, possibly reflecting known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism. The identification of invertebrate orthologs of human MTP may enable structural and functional dissection of the multiple roles of human MTP in apoB assembly as well as apoB-independent effects on intracellular lipid trafficking (23Wang Y. Tran K. Yao Z. J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 24Hamilton R.L. Wong J.S. Cham C.M. Nielsen L.B. Young S.G. J. Lipid Res. 1998; 39: 1543-1557Abstract Full Text Full Text PDF PubMed Google Scholar, 25Kilinski A. Rustaeus S. Vance J.E. J. Biol. Chem. 2002; 277: 31516-31525Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Sequence Alignments—MTP sequences were aligned with the ClustalW multiple sequence alignment tool using the BLOSUM 30 matrix as implemented in MacVector 7.1.1 (Accelrys, Inc.). Sequence records used were as follows: Homo sapiens (human): locus, MTP_HUMAN; GenBank™ accession number, P55157. Bos taurus (bovine): locus, A46764; accession number, A4764. Mus musculus (mouse) MTP: locus, Mttp; accession, NP_032668, Fugu rubripes (puffer fish): Copyright definition, SINFRUP00000088005, D. melanogaster (fruit fly): locus, CG9342; accession number, NP_610075. Anopheles gambiae (mosquito): locus, EAA13951; accession number, EAA14951. Signal peptidase cleavage sites for the Fugu and Drosophila sequences were predicted using the SignalIP prediction algorithm (26Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4934) Google Scholar) as implemented by the SignalIP V2.0 server on the web. The amino terminus of mature human MTP was based on data reported by Shoulders et al. (27Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M.E. Jarmuz A. Grantham T.T. Leoni P.R.D. 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). The full-length EST for D. melanogaster CG9342 cloned into the vector, pOT2, was obtained from ResGen (Invitrogen). For expression in COS cells, the CG9342 cDNA was transferred to expression vector pCMV5 (28Andersson S. Davis D.L. Dahlbäack H. Jörnvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). ApoB34H (amino-terminal 34% of apoB with a carboxyl-terminal His6 tag) and apoB34F (amino-terminal 34% of apoB with a carboxyl-terminal FLAG tag) were constructed by overlap PCR extension and cloned into pCMV5 as described previously for apoB41F (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). Cell Culture and Metabolic Radiolabeling—COS-1 cells were grown in DMEM (Media Tech) as described previously (30Shelness G.S. Morris-Rogers K.C. Ingram M.F. J. Biol. Chem. 1994; 269: 9310-9318Abstract Full Text PDF PubMed Google Scholar) and transfected at 50 – 60% confluence using the Fugene-6 transfection reagent (Roche Applied Science) (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar) or, as noted, by the DEAE-dextran method (31Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar). For coexpression, cells were transfected with the indicated apoB construct and human MTP 97-kDa subunit (32Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M.-E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Nature. 1993; 365: 65-69Crossref PubMed Scopus (404) Google Scholar), Drosophila CG9342, or truncated human placental alkaline phosphatase (AP) at a mass ratio of 2:1. Twenty-four hours after transfection, cells were radiolabeled with 100 μCi/ml [35S]Met/Cys (EasyTag Express Protein Labeling Mix, PerkinElmer Life Sciences) in Met/Cys-deficient DMEM (ICN Biomedicals) for the indicated times. Following labeling, cell media and lysates were immunoprecipitated with anti-apoB polyclonal antibodies as described previously (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). MTP Inhibition—The MTP inhibitors BMS-200150 and BMS-197636 (Bristol-Myers Squibb) (33Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson Jr., J.K. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek Jr., C.P. Biller S.A. Gregg R.E. Wetterau J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11991-11995Crossref PubMed Scopus (144) Google Scholar, 34Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C.H. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S.J. Maccagnan T.J. Ricci B. Yan M.J. Young D. Chen Y. Fryszman O.M. Logan J.V.H. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Science. 1998; 282: 751-754Crossref PubMed Scopus (251) Google Scholar) were dissolved in Me2SO at concentrations of 10 and 2 mm, respectively. Final Me2SO concentrations were normalized in each experimental set of dishes and did not exceed 0.5%. Twenty-four hours after transfection, cells were washed with PBS and incubated for 30 min with Met/Cys-deficient DMEM containing the indicated concentration of inhibitor. After removal of preincubation media, fresh media was added containing inhibitor and 100 μCi/ml [35S]Met/Cys. After 5 h, cell lysate and media samples were immunoprecipitated with anti-apoB antibodies and analyzed by 6% SDS-PAGE (apoB34 and apoB41) or 12.5% SDS-PAGE (apoB6.6). The effect of MTP inhibition on apoB41F mass secretion was analyzed by ELISA as described previously (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). Density Gradient Centrifugation—Density analysis of apoB34H-containing lipoprotein particles from human MTP- or CG9342-transfected cells was performed as described (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). After centrifugation, twelve 1-ml samples were collected and concentrated to ∼50 μl using Centricon 30 centrifugal concentrators (Millipore). Samples were diluted with 1 ml of lysis buffer (1% Triton X-100, 300 mm NaCl, 25 mm Tris-HCl, pH 7.4, 1 mm PMSF) and subjected to immunoprecipitation. Samples were then analyzed by 6% SDS-PAGE and fluorography. Each gradient is representative of media collected from one 150-mm dish of transfected cells. Oleate Labeling and Affinity Purification of ApoB34H-containing Lipoproteins—COS-1 cells in 150-mm dishes were transfected with 10 μg of apoB34H and 5 μg of either human MTP or CG9342 by the DEAE-dextran method. Forty hours post-transfection, cells were washed twice with PBS. Radiolabeling was performed in a 4:1 mixture of Met/Cys-deficient DMEM and complete DMEM containing 0.5% fatty acid free bovine serum albumin (Sigma), 10 μCi/ml [3H]oleate (PerkinElmer Life Sciences), and 20 μCi/ml [35S]Met/Cys. After labeling for 24 h, media was clarified by centrifugation at 1000 × g for 5 min and adjusted to 150 mm NaCl (final NaCl concentration, ∼300 mm), 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mm PMSF. ApoB34H was then affinity-purified from media by addition of a 375-μl bed volume of nickel-nitrilotriacetic acid-agarose (Qiagen) and incubation for 90 min at 4 °C with inversion. The suspension was passed through a 15-ml polypropylene disposable column (Bio-Rad), and the column was washed twice with 15 ml of wash buffer I (50 mm NaH2PO4, 300 mm NaCl, and 5 mm imidazole, pH 8.0) and twice with 15 ml of wash buffer II (50 mm NaH2PO4, 300 mm NaCl, pH 8.0). The column was eluted three times with 0.75 ml of elution buffer (50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, pH 8.0). Eluted fractions were then pooled and concentrated to ∼100 μl using a Centricon-30 centrifugal concentrator. Ten percent of each eluted fraction was reserved for analysis by SDS-PAGE and fluorography to verify protein purity. Lipid Composition of Affinity Purified Lipoproteins—Lipids were extracted from affinity-purified lipoproteins by the method of Bligh and Dyer (35Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). Briefly, samples were adjusted to 0.5 ml with PBS followed by addition of 1.88 ml of chloroform:methanol (1:2, v/v). Ten microliters of a mixture of 5 mg/ml egg yolk lecithin, triolein, dioleoyl glycerol, and cholesteryl oleate (in chloroform) and 0.626 ml of chloroform was added, followed by brief vortexing. Samples were acidified by addition of 0.626 ml of 0.05% concentrated sulfuric acid, again followed by brief vortexing. After centrifugation at 1500 rpm for 10 min at room temperature, the upper phase was removed by aspiration and the lower phase was dried at 45 °C under a stream of nitrogen. The sides of the tube were rinsed with 200 μl of chloroform and again dried. Samples were redissolved in 50 μl of chloroform and applied to a polyester-backed silica gel TLC plate (Whatman, PE SIL G). Plates were developed first in a polar solvent of chloroform:methanol:acetic acid:water (65:45:12:6, v/v), by running the solvent front ∼1/3 up the length of the plate. After air drying, plates were developed in a neutral solvent tank containing hexane:ether:acetic acid (80:20:1, v/v). Lipid standards were visualized by incubation in iodine vapor, and areas containing phosphatidylcholine, diacylglycerol, triacylglycerol, and cholesteryl ester were cut and quantified by liquid scintillation counting. Control experiments, in which the entire length of the TLC plate was cut and counted, revealed that these were the only radiolabeled lipid species present above background. Alignment of CG9342 with Vertebrate MTPs—The deduced amino acid sequence of CG9342 was compared with human and Fugu MTP (Fig. 1). The human and lower vertebrate (Fugu) sequences shared 55% identity/72% similarity over a 882-amino acid alignment, with one gaped region. The Drosophila sequence displayed 23% identity/42% similarity with human MTP and 22% identity/41% similarity with Fugu MTP, each over a 902-amino acid alignment containing 22 gapped regions. Hence, the identity between higher and lower vertebrate MTP is substantial, suggesting analogous functional roles. However, the considerably lower identity between the insect and vertebrate sequences, coupled with different lipid transport mechanisms observed across these species (16Canavoso L.E. Jouni Z.E. Karnas K.J. Pennington J.E. Wells M.A. Annu. Rev. Nutr. 2001; 21: 23-46Crossref PubMed Scopus (464) Google Scholar), makes the assignment of Drosophila CG9342 function difficult based on sequence comparison alone. CG9342 Induces the Secretion of ApoB—A functional hallmark of MTP is its capacity to induce the secretion of apoB (36Leiper J.M. Bayliss J.D. Pease R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar, 37Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R.E. Wetterau J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7628-7632Crossref PubMed Scopus (189) Google Scholar, 38Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z.M. Talmud P. Attie A.D. J. Biol. Chem. 1996; 271: 8682-8691Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). To explore whether CG9342 encodes a functional MTP, it was transiently expressed along with human apoB34 in COS cells. ApoB34 is incapable of undergoing appreciable secretion into media (M) in the absence of MTP (Fig. 2, lane 2). Cotransfection with human MTP dramatically induced apoB34 secretion (lane 4). CG9342 also induced apoB34 secretion, although with slightly lower efficiency (lanes 6 and 8). Another hallmark of MTP is its capacity to interact physically with apoB (10Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Köchl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar, 39Wu X.J. Zhou M.Y. Huang L.S. Wetterau J. Ginsberg H.N. J. Biol. Chem. 1996; 271: 10277-10281Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 40Patel S.B. Grundy S.M. J. Biol. Chem. 1996; 271: 18686-18694Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 41Hussain M.M. Bakillah A. Nayak N. Shelness G.S. J. Biol. Chem. 1998; 273: 25612-25615Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). As observed in immunoprecipitates of cell extracts (C), both human MTP (lane 3) and CG9342 (lanes 5 and 7) appear to coimmunoprecipitate with apoB34. It has been known for some time that human MTP migrates more rapidly during SDS-PAGE than predicted from its calculated molecular weight (5Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar), which may explain the relatively slower migration of Drosophila CG9342. CG9342 Promotes the Secretion of ApoB34 as a Buoyant Lipoprotein Particle with a Density and Lipid Composition Similar to That Produced by Human MTP—To directly assess whether the Drosophila protein is capable of assembling apoB34 with lipid, apoB34 was cotransfected into COS cells with either human MTP or CG9342. After radiolabeling with [35S]Met/Cys, media was subjected to density gradient centrifugation and apoB was recovered by immunoprecipitation. ApoB34 displayed a relatively heterogeneous density distribution profile with a peak of ∼1.169 g/ml (Fig. 3). This value is in agreement with density values obtained by others in stably transfected McA-RH7777 rat hepatoma cells (42Yao Z. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar). The composition of lipids associated with apoB34 was explored by radiolabeling transfected cells with [3H]oleate. After affinity purification of the apoB34-containing particles, lipids were separated by TLC and quantified by liquid scintillation counting. As observed in Fig. 4, the relative composition of [3H]oleate-labeled lipids associated with apoB34-containing particles secreted from COS cells cotransfected with either human MTP or Drosophila CG9342 was virtually identical.Fig. 4ApoB34-containing particles formed by human MTP or CG9342 display similar lipid compositions. COS-1 cells in 150-mm dishes were transfected by the DEAE-dextran method with apoB34H and either human MTP (hMTP, hatched bars) or CG9342 (unshaded bars). Forty hours after transfection, cells were labeled with [35S]Met/Cys and [3H]oleate for 24 h. ApoB34H was purified from media by nickel chromatography and [3H]oleate-labeled phosphatidylcholine (PC), diacylglycerol (DG), triacylglycerol (TG), and cholesteryl ester (CE) were quantified by TLC and liquid scintillation counting. The values from duplicate samples, each consisting of particles purified from two, 150-mm dishes, were used to calculate an average percent radiolabeled lipid composition for the apoB34-containing lipoproteins secreted by human MTP- and CG9342-cotransfected cells. Error bars, where visible, depict the data range.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CG9342 and Human MTP Display Differential Susceptibilities to Chemical Inhibitors of Vertebrate MTP—To further explore whether the CG9342 gene product displays properties consistent with the behavior of vertebrate MTP, we tested its response to two well characterized MTP inhibitors. Cells were cotransfected with apoB41 or apoB6.6 and either AP, human MTP, or CG9342. ApoB41 was used in these studies because, unlike apoB34, which produces a very low level of background secretion (Fig. 2, lane 2), no detectable background secretion of apoB41 is observed in the absence of MTP (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar, 36Leiper J.M. Bayliss J.D. Pease R.J. Brett D.J. Scott J. Shoulders C.C. J. Biol. Chem. 1994; 269: 21951-21954Abstract Full Text PDF PubMed Google Scholar). ApoB6.6, whose secretion is MTP-independent, served as a control for nonspecific effects of the inhibitors (29Sellers J.A. Shelness G.S. J. Lipid Res. 2001; 42: 1897-1904Abstract Full Text Full Text PDF PubMed Google Scholar). Twenty-four hours after transfection, cells were radiolabeled with [35S]Met/Cys in the presence of 0–20 μm BMS-200150 for 5 h (33Jamil H. Gordon D.A. Eustice D.C. Brooks C.M. Dickson Jr., J.K. Chen Y. Ricci B. Chu C.H. Harrity T.W. Ciosek Jr., C.P. Biller S.A. Gregg R.E. Wetterau J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93:

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