The Pancreatitis-induced Vacuole Membrane Protein 1 Triggers Autophagy in Mammalian Cells
2007; Elsevier BV; Volume: 282; Issue: 51 Linguagem: Inglês
10.1074/jbc.m706956200
ISSN1083-351X
AutoresAlejandro Ropolo, Daniel Grasso, Romina Pardo, Maria Luisa Sacchetti, Cendrine Archange, Andrea Lo Ré, Mylène Seux, Jonathan A. Nowak, Claudio González, Juan Iovanna, María I. Vaccaro,
Tópico(s)Pancreatitis Pathology and Treatment
ResumoAutophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process. Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process. Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells (1Codogno P. Meijer A.J. Cell Death Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (946) Google Scholar, 2Kroemer G. Jäättelä M. Nat. Rev. Cancer. 2005; 5: 886-897Crossref PubMed Scopus (1037) Google Scholar, 3Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3189) Google Scholar). 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Klöppel G. Kern H.F. Adler G. Virchows Arch. A Pathol. Anat. Histopathol. 1989; 415: 115-123Crossref PubMed Scopus (46) Google Scholar). Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes, which eventually acquire lysosomal-like features (13Mizushima N. Cell Death Differ. 2005; 12: 1535-1541Crossref PubMed Scopus (404) Google Scholar, 14Klionsky D.L. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2969) Google Scholar). Autophagy is mediated by a set of evolutionarily conserved gene products (termed the Atg proteins) originally discovered in yeast (15Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar). In mammalian cells, Beclin 1 (3Levine B. Klionsky D.J. Dev. 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Cell. 2005; 122: 927-939Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar) promotes autophagosome formation when it functions as part of a complex with the Class III phosphatidylinositol 3-kinase (PI3K) 6The abbreviations used are:PI3Kphosphatidylinositol 3-kinaseVMP1vacuole membrane protein 1LC3light-chain 3siRNAsmall interfering RNA3-MA3-methyladenineGSTglutathione S-transferaseGFPgreen fluorescent proteinmTORmammalian target of rapamycinAtgautophagy-related domainRFPred fluorescent protein. 6The abbreviations used are:PI3Kphosphatidylinositol 3-kinaseVMP1vacuole membrane protein 1LC3light-chain 3siRNAsmall interfering RNA3-MA3-methyladenineGSTglutathione S-transferaseGFPgreen fluorescent proteinmTORmammalian target of rapamycinAtgautophagy-related domainRFPred fluorescent protein. mediating the localization of other autophagic proteins to the autophagosomal membrane (19Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (804) Google Scholar). However, despite the advances in understanding autophagy, autophagosome formation in mammalian cells is a complex process, and neither the molecular mechanisms nor all the implicated genes involved in its formation are fully elucidated (20Juhasz G. Neufeld T.P. PLoS Biol. 2006; 4: e36Crossref PubMed Scopus (122) Google Scholar, 21Klionsky D.J. J. Cell Sci. 2005; 118: 7-18Crossref PubMed Scopus (766) Google Scholar). phosphatidylinositol 3-kinase vacuole membrane protein 1 light-chain 3 small interfering RNA 3-methyladenine glutathione S-transferase green fluorescent protein mammalian target of rapamycin autophagy-related domain red fluorescent protein. phosphatidylinositol 3-kinase vacuole membrane protein 1 light-chain 3 small interfering RNA 3-methyladenine glutathione S-transferase green fluorescent protein mammalian target of rapamycin autophagy-related domain red fluorescent protein. The pancreatitis-associated protein named vacuole membrane protein 1 (VMP1) is a transmembrane protein with no known homologues in yeast. We identified the VMP1 transcript because it is highly activated in acinar cells early during experimental acute pancreatitis and its transient expression induced the formation of numerous cytoplasmic vacuoles in mammalian cells (22Dusetti N.J. Jiang Y. Vaccaro M.I. Tomasini R. Samir Azizi A. Calvo E.L. Ropolo A. Fiedler F. Mallo G.V. Dagorn J.C. Iovanna J.L. Biochem. Biophys. Res. Commun. 2002; 290: 641-649Crossref PubMed Scopus (76) Google Scholar). VMP1 mRNA in situ expression correlated with the formation of vacuoles in acinar cells during experimental acute pancreatitis and in the course of chronic pancreatitis in WBN/Kob rats (23Vaccaro M.I. Grasso D. Ropolo A. Iovanna J.L. Cerquetti M.C. Pancreatology. 2003; 3: 69-74Crossref PubMed Scopus (34) Google Scholar, 24Jiang P.H. Motoo Y. Vaccaro M.I. Iovanna J.L. Okada G. Sawabu N. Pancreas. 2004; 29: 225-230Crossref PubMed Scopus (17) Google Scholar). In this study we tested the hypothesis that VMP1 expression is involved in the autophagic process. We show that VMP1 is a novel autophagy-related membrane protein that triggers autophagosome formation in mammalian cells. VMP1 is induced by autophagy stimuli; it is required for autophagosome development and its expression triggers autophagy, even under nutrient-replete conditions. VMP1 interacts with Beclin 1 through its hydrophilic C-terminal region, which we named Atg domain because it is essential for autophagosome formation. We also show that VMP1 co-localizes with the microtubule-associated protein 1 light chain 3 (LC3) in vacuole membranes in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, we found that VMP1 expression promotes the formation of LC3-positive vacuoles when specifically targeted to the pancreas acinar cells in transgenic mice. Mammalian Cell Lines, Transfections, and Treatments—Human HeLa, 293T and MCF7, and mouse NIH3T3 (ATCC) cell lines were used. Low-Beclin 1 levels in MCF7 cells were verified by Western blot. Cells were transfected using FuGENE-6 reagent (Roche Applied Science). Plasmids were pcDNA4-V5-His (Invitrogen) and pEGFP-N1 (Clontech), containing full-length rat VMP1 cDNA (NM_138839) (22Dusetti N.J. Jiang Y. Vaccaro M.I. Tomasini R. Samir Azizi A. Calvo E.L. Ropolo A. Fiedler F. Mallo G.V. Dagorn J.C. Iovanna J.L. Biochem. Biophys. Res. Commun. 2002; 290: 641-649Crossref PubMed Scopus (76) Google Scholar) or VMP1ΔAtg subcloned within the HindIII and BamHI restriction sites. Full-length human Beclin 1 cDNA (NM_003766) was subcloned into pECFP-C1 (Clontech) within the EcoRI and SalI or into pcDNA3 (Invitrogen) within EcoRI and ApaI restriction sites. pRFP-C1 containing full-length rat LC3 was kindly provided by Dr. Maria I. Colombo (Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina). Cells were also transfected using Oligofectamine (Invitrogen)-mediated transfer with siRNA pre-designed for human VMP1 mRNA using 5′-GGCAGAAUAUUGUCCUGUGtt-3′, as sense, and 5′-CACAGGACAAUAUUCUCUGCCtt-3′, as antisense (Ambion ID32935) and 24 h later, cells were subjected to autophagy stimuli for an additional 6-h period. For autophagy inhibition, cells were treated with 10 mm 3-methyladenine (3-MA) (Sigma) 2 h before and during co-transfection with pcDNA4-VMP1 and pRFP-LC3. The efficacy of 3-MA treatment was verified on starved cells (data not shown). For lysosomal hydrolase inhibition, cells were treated with E64d (10 μg/ml) for 4 h before processed. Autophagy was induced by amino acid/serum-deprived medium using Earle's balanced salt solution (Invitrogen) or 55 μm rapamycin (Calbiochem) treatments. Fluorescence was observed using a fluorescence microscope Nikon Eclypse 200 (Plan100×), an inverted fluorescence microscopy Olympus GX71, a confocal microscope Nikon Eclipse C1 (Plan40×/0.95 and Plan60×/1.40), or an inverted confocal microscope Olympus FV1000 (PLAPO N/1.42). Transmission Electron Microscopy—Cells were fixed without being brought into suspension and processed for transmission electron microscopy by standard procedures. Grids were examined under a Carl Zeiss C-10 electron microscope (Laboratorio Nacional de Investigación y Servicios en Microscopía Electrónica, University of Buenos Aires). Percentage of RFP-LC3 Cells with Punctate Staining—The number of cells with punctate staining per 100 fluorescent RFP-LC3 transfected cells was determined in three independent experiments. To quantify, the number of fluorescent cells with punctate staining was counted in six random fields representing 100 fluorescent cells and expressed as the mean ± S.D. of combined results. We consider an RFP-LC3 cell to have punctate staining when all the red fluorescence is present as punctate and no diffused protein remains. Recombinant GST-VMP1 Peptides and His6-Beclin 1 Protein—VMP1 hydrophilic peptides were amplified from pcDNA4-VMP1 by PCR and subcloned into pGEX-5X-2 (Amersham Biosciences) to obtain GST-VMP1 peptides. Peptides were expressed in Escherichia coli BL21 and purified by glutathione affinity chromatography (Amersham Biosciences). Beclin 1 cDNA was obtained from HeLa cell total RNA extracts and cloned into pQE31 (Qiagen). His6-Beclin 1 was expressed in E. coli M15 and purified using nickel-nitrilotriacetic acid-agarose beads (Qiagen). Membrane Isolation—Cells were washed and homogenized with a motor driven Teflon pestle homogenizer in ice-cold SBE buffer (250 mm sucrose, 1 mm EGTA, 10 mm Hepes/KOH, pH 7.5) containing protease inhibitors. Homogenates were centrifuged twice at 500 × g for 15 min. The resulting supernatants were centrifuged at 100,000 × g for 1 h. Membrane pellets were resuspended in ice-cold SBE buffer and treated with 1% Nonidet P-40 (Nonidet P-40), 0.2 m Na2CO3, pH 11.0, or 1.5 m NaCl. All steps were performed at 4 °C. The membrane pellets were obtained by centrifugation, and retained proteins were separated by SDS-PAGE. Western blot analysis was performed using as secondary antibody a peroxidase-labeled IgG antibody provided with the ECL-kit (Amersham Biosciences). Immunoblotting was performed using the ECL kit and membranes were exposed to a Kodak BioMax film. Pulldown Assays—HeLa cells transfected with the pcDNA4-VMP1, pcDNA4-VMP1ΔAtg, and pcDNA4-empty plasmids were lysed by sonication and solubilized with 1% Triton X-100, followed by a 30-min centrifugation at 100,000 × g. The supernatants containing His-tagged proteins were incubated with nickel-nitrilotriacetic acid-agarose beads (Qiagen) for 2 h at 4 °C, extensively washed with a buffer containing 5 mm imidazole, and eluted with a buffer containing 120 mm imidazole. Proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. In another experiment His6-Beclin 1 produced in E. coli was incubated in nickel-nitrilotriacetic acid-agarose beads with the purified GST-VMP1 peptides. Co-immunoprecipitation Assays—For the co-immunoprecipitation assays, the supernatant from lysates of pcDNA4-VMP1-transfected or rapamycin-treated cells were incubated with antisera bound to protein A-Sepharose™ Cl-4B beads (Amersham Biosciences) for 2 h at 4 °C and washed extensively. Bound proteins were eluted with 100 mm glycine-HCl, pH 2.5, precipitated by 5% trichloroacetic acid, washed with ice-cold acetone, and resuspended in a SDS sample buffer. Antibodies—Polyclonal rabbit antiserum to VMP1 against the peptide MAQSYAKRIQQRLNSEEKTK (residues 386-406) was obtained and used at 1:100 dilution. Polyclonal goat anti-LC3, goat anti-Beclin 1, rabbit anti-GST, monoclonal mouse anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal mouse anti-V5 (Invitrogen) antibodies were used according manufacturer. Donkey anti-rabbit and anti-goat Alexa Fluor 488 and 590, and rabbit anti-mouse Alexa Fluor 590 (Molecular Probes) antibodies were used for immunofluorescence. Peroxidase-labeled anti-rabbit, anti-mouse, and anti-goat IgG antibodies were used for Western blot according Amersham Biosciences. Caerulein-induced Pancreatitis—Male Wistar rats weighing 200-250 g were used. Animals were housed with free access to food and water. Experiments were performed according to the standard ethical and legal guidelines of the Administración Nacional de Medicamentos, Alimentos y Tecnología Medica, University of Buenos Aires, and the European Union regulations for animal experiments. Pancreatitis was induced by seven intraperitoneal injections of caerulein (Sigma, 50 μg/kg) given at 1-h intervals; the rats were killed by decapitation at different times after the first injection. In one series of experiments, the pancreas were removed, homogenized in HEPES buffer, pH 7.4, containing protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin, 5 μg/ml leupeptin, and 2.5 μg/ml aprotinin) and processed for Western blot analysis. In another series of experiments, the pancreas were removed, fixed in formalin buffer, and processed for immunofluorescence. Transgenic Mice—The transgene cassette was made using the pBEG vector (25Fernandez-Zapico M.E. Mladek A. Ellenrieder V. Folch-Puy E. Millar L. Urrutia R. EMBO J. 2003; 22: 4748-4758Crossref PubMed Scopus (86) Google Scholar). The expression cassette contains the acinar-specific control region (-500 to +8) from the rat elastase I gene and the human growth hormone 3′-untranslated region (+500 to +2657). This construct was digested with BamHI, filled in, dephosphorylated, and ligated with rat VMP1-EGFP released from pEGFP-VMP1 plasmid. A 1940-kb HindIII/NotI fragment was isolated and used for microinjections into in-bred Friend virus B-type susceptibility zygotes. Genomic DNA was prepared and tested by Southern blot or PCR. VMP1 Expression Triggers Autophagy—To know if VMP1 triggers autophagy, we transfected HeLa cells with the expression plasmid pcDNA4-VMP1, which codes for the VMP1-V5 fusion protein. The pcDNA4-empty plasmid was used in controls. Cells were cultured in nutrient and grown factor-replete conditions and fixed in glutaraldehyde 24 h later to perform transmission electronic microscopy. We found that cells expressing VMP1 showed multiple autophagic features. Fig. 1A shows cup-shaped structures, double-membrane structures containing cytoplasmic material (autophagosome like structure), as well as single-membrane structures containing cytoplasmic constituents at different stages of degradation (autolysosome-like structure) (26Gozuacik D. Kimchi A. Oncogene. 2004; 23: 2891-2906Crossref PubMed Scopus (1254) Google Scholar, 27Klionsky D.J. Nature. 2004; 431: 31-32Crossref PubMed Scopus (79) Google Scholar). The same morphological features were obtained when 293T cells were transfected with the VMP1-expression plasmid, and they did not differ from those obtained in rapamycin-treated cells (data not shown). During autophagy, the cytosolic form of LC3 (LC3-I) undergoes C-terminal proteolytic and lipid modifications (LC3-II) and translocates from the cytosol to the autophagosomal membrane (28Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar, 29Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1923) Google Scholar). LC3 is currently used as a specific marker of autophagy (30Yoshimori T. Biochem. Biophys. Res. Commun. 2004; 313: 453-458Crossref PubMed Scopus (462) Google Scholar, 31Klionsky D.J. Cuervo A.M. Seglen P.O. Autophagy. 2007; 3: 181-206Crossref PubMed Scopus (557) Google Scholar). To confirm the extent and specificity of VMP1 autophagosome induction, we first immunostained pcDNA4-VMP1-transfected 293T cells with a specific LC3 antibody, and we observed the recruitment of endogenous LC3 in punctate structures consistent with autophagy (Fig. 1B). Then, we investigated LC3-I and -II forms by Western blot (Fig. 1C). We found induction of LC3 with increased LC3-II form signal in pcDNA4-VMP1-transfected cells (Fig. 1C, lane 1). Because intra-autophagosomal LC3-II is degraded by lysosomal proteases, we blocked its proteolysis using the lysosomal protease inhibitor E64d (32Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (936) Google Scholar), and we found that LC3-II signal was enhanced in VMP1-expressing cells (Fig. 1C, lane 4). In another series of experiments HeLa, 293T, and NIH3T3 cells cultured under nutrient and grown factor-replete conditions were concomitantly transfected with an expression plasmid encoding for the RFP-LC3 fusion protein and pcDNA4-VMP1 or pcDNA4-empty plasmids. Fig. 1D shows the recruitment of LC3 fusion protein in punctate structures in VMP1-transfected cells in contrast to the diffuse RFP-LC3 fusion protein signal observed in control cells. We determine the percentage of fluorescent RFP-LC3 cells with punctate staining in three independent experiments per cell line as under “Experimental Procedures.” We found high recruitment of LC3 in VMP1-transfected cells. Finally, we investigated the potential for inhibiting the pathway with an agent well documented to inhibit autophagy. The progression of the autophagy is sensitive to the PI3K inhibitors such as 3-MA, with the target being the Class III PI3K (33Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). We treated cells with 3-MA before the co-transfection with pRFP-LC3 and pcDNA4-VMP1 expression plasmids, and we found that the percentage of RFP-LC3 cells with punctate staining was low and almost the same as the observed in pcDNA4-empty transfected cells (Fig. 1D). These results collectively demonstrate that VMP1 expression triggers autophagy in mammalian cells, even under nutrient-replete conditions. VMP1 Expression Is Required for Extracellular Stimuli-induced Autophagy—The autophagy trafficking pathway was first described as a cellular adaptation to starvation (34Mitchener J.S. Shelburne J.D. Bradford W.D. Hawkins H.K. Am. J. Pathol. 1976; 83: 485-491PubMed Google Scholar). To investigate whether starvation activates endogenous VMP1 expression, we developed a rabbit polyclonal anti-VMP1 antibody. We subjected HeLa cells to a standard starvation protocol (amino acid/serum-deprived medium) and then analyzed the time course of VMP1-mRNA expression by reverse transcription-PCR (Fig. 2A) and of VMP1-protein expression by Western blot (Fig. 2B). VMP1 expression was activated under starvation, and it was evident after 2-h treatment. Moreover, starvation-induced VMP1 expression was detected in punctate structures by immunofluorescence, whereas it was almost not detectable under nutrient-replete conditions (Fig. 2C). mTOR kinase plays a central role in the amino acid pool-sensing mechanism. In response to starvation, mTOR is inhibited, resulting in the induction of autophagy (35Jacinto E. Hall M.N. Nat. Rev. Mol. Cell. Biol. 2003; 4: 117-126Crossref PubMed Scopus (513) Google Scholar). Because mTOR can be inhibited by rapamycin, we treated HeLa cells with rapamycin as a pharmacological agent to induce autophagy. We found that mTOR inhibition induces VMP1-mRNA and VMP1-protein expression (Fig. 2, A-C). To establish whether VMP1 is required for autophagy, we reduced the expression of VMP1 using the small interfering RNA (siRNA) strategy. HeLa cells were transfected with VMP1-siRNA and then subjected to standard starvation protocol or rapamycin treatment. VMP1 expression was efficiently knocked down (Fig. 2, D and E). We found that autophagosome formation was almost completely inhibited in VMP1-siRNA cells under both treatments, as evidenced by the distribution of the RFP-LC3 fluorescence fusion protein (Fig. 2F). We analyzed three independent experiments per treatment, and we found that the percentage of RFP-LC3 cells with punctate staining in VMP1-siRNA transfected cells was highly reduced in comparison with those transfected with scramble siRNA (Fig. 2F). These findings show that VMP1 expression is required for extracellular stimuli-induced autophagy. VMP1 Remains as a Membrane Protein during Autophagy—VMP1 primary structure predicts a transmembrane protein (22Dusetti N.J. Jiang Y. Vaccaro M.I. Tomasini R. Samir Azizi A. Calvo E.L. Ropolo A. Fiedler F. Mallo G.V. Dagorn J.C. Iovanna J.L. Biochem. Biophys. Res. Commun. 2002; 290: 641-649Crossref PubMed Scopus (76) Google Scholar). GFP moiety folds properly and becomes fluorescent only if a membrane protein is stably inserted into the membrane (36Wagner S. Bader M.L. Drew D. de Gier J.W. Trends Biotechnol. 2006; 24: 364-371Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). To verify if VMP1 remains in membrane structure we transfected cells with pEGFP-VMP1 expression plasmid, and cells were cultured in nutrient and grown factor-replete conditions. After 24 h the GFP fluorescence of the fusion protein VMP1-EGFP was observed in vacuole membranes induced by its own expression (Fig. 3A). We then analyzed whether VMP1 co-localizes with endogenous LC3 in the VMP1-induced vacuoles. We performed immunofluorescence using the anti-LC3 and anti-V5 antibodies in HeLa cells transfected with VMP1-V5 expression plasmid. We found a remarkable co-localization between VMP1-V5 fusion protein and endogenous LC3 in VMP1-induced vacuoles (Fig. 3B). To know if the endogenous VMP1 also remains as a membrane protein during autophagy, we performed subcellular fractioning of HeLa cells undergoing rapamycin-induced autophagy and investigated VMP1 in membrane preparations by Western blot. Fig. 3C shows that, although the protein is not detectable in untreated cells, VMP1 is found in the membrane preparations from cells undergoing autophagy, and the signal persists when membrane fractions are treated with 1.5 m NaCl or exposed to pH 11.0. These results indicate that VMP1 functions as a membrane protein during autophagy. VMP1 Is a Beclin 1-binding Protein—To obtain a mechanistic insight as to how VMP1 triggers autophagy, we analyzed its function in the molecular pathway of the autophagosome formation. During autophagy Beclin 1-Class III PI3K complex (37Kihara A. Kabeya Y. Ohsumi Y. Yoshimori T. EMBO Rep. 2001; 2: 330-335Crossref PubMed Scopus (724) Google Scholar) is thought to undergo subcellular distribution to a membrane structure, which eventually leads to the recruitment of autophagy proteins and the proper conjugation of LC3 to membrane phospholipids (19Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (804) Google Scholar, 38Mizushima N. Yamamoto A. Hatano M. Koboyashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1158) Google Scholar). However, the transmembrane protein with which Beclin 1 complex interacts remains elusive. To investigate if VMP1 is a target membrane protein with which Beclin 1 interacts, we first analyzed if Beclin 1 localizes in the membrane of the VMP1-induced vacuoles. We concomitantly transfected 293T cells with pEGFP-VMP1, pRFP-LC3, and pECFP-Beclin 1 expression plasmids (results are shown in Fig. 4A). We found a remarkable co-localization between VMP1, LC3, and Beclin 1 fluorescent fusion proteins, suggesting that Beclin 1 could attach to VMP1-induced vacuole membranes. Then, we studied if VMP1 interacts with Beclin 1. We performed a pulldown assay using the VMP1-V5-His6 fusion protein. Lysates prepared from HeLa cells transfected with pcDNA4-VMP1 were treated with Triton X-100 and incubated with nickel-agarose beads. After extensively washed, retained proteins were eluted with imidazole and separated by SDS-PAGE followed by anti-Beclin 1 or anti-V5 antibody immunoblotting. VMP1-V5 and Beclin 1 were found in eluates (Fig. 4B). Control experiments showed that neither Beclin 1 nor VMP1-V5 was detected in the elution fraction of pcDNA4-empty-transfected cells. In another series of experiments, lysates from pcDNA4-VMP1-transfected cells were incubated with the anti-Beclin 1 antibody. Controls were incubated with control serum. Immunoprecipitates were separated and immunoblotted for VMP1-V5 with anti-V5 antibody. VMP1-V5 was detected in Beclin 1 antibody-treated lysates but not in the controls (Fig. 4C). To analyze the physiological relevance of the VMP1-Beclin 1 interaction, we investigated whether endogenous VMP1 interacts with endogenous Beclin 1 in cells developing rapamycin-induced autophagy by co-immunoprecipitation experiments. Endogenous Beclin 1 an
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