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• W2023198417 abstract "Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p). Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p). Phosphoinositides are phosphorylated derivatives of phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; EGF, epidermal growth factor; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; M6PR, mannose 6-phosphate receptor; MVB, multivesicular body; ORF, open reading frame; P2, bisphosphate; PH, pleckstrin homology; VPS, vacuolar protein sorting; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PBS, phosphate-buffered saline; TGN, trans Golgi network; CHMPs, charged multivesicular body proteins.1The abbreviations used are: PtdIns, phosphatidylinositol; EGF, epidermal growth factor; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; M6PR, mannose 6-phosphate receptor; MVB, multivesicular body; ORF, open reading frame; P2, bisphosphate; PH, pleckstrin homology; VPS, vacuolar protein sorting; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PBS, phosphate-buffered saline; TGN, trans Golgi network; CHMPs, charged multivesicular body proteins. that have been implicated in diverse cellular functions such as cell growth, apoptosis, transcription, cytoskeletal organization, and membrane trafficking (1Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1357) Google Scholar, 2Corvera S. D'Arrigo A. Stenmark H. Curr. Opin. Cell Biol. 1999; 11: 460-465Crossref PubMed Scopus (184) Google Scholar). The most recent addition to the repertoire of phosphoinositides found in eukaryotic cell membranes came with the discovery of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) in yeast and fibroblasts (3Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (389) Google Scholar, 4Whiteford C.C. Brearley C.A. Ulug E.T. Biochem. J. 1997; 323: 597-601Crossref PubMed Scopus (127) Google Scholar). PtdIns(3,5)P2 is synthesized by the PtdIns-3-phosphate 5-kinase Fab1p and is involved in regulating vacuolar homeostasis in yeast (5Cooke F.T. Dove S.K. McEwen R.K. Painter G. Holmes A.B. Hall M.N. Michell R.H. Parker P.J. Curr. Biol. 1998; 8: 1219-1222Abstract Full Text Full Text PDF PubMed Google Scholar, 6Gary J.D. Wurmser A.E. Bonangelino C.J. Weisman L.S. Emr S.D. J. Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (339) Google Scholar, 7Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 8Dove S.K. McEwen R.K. Mayes A. Hughes D.C. Beggs J.D. Michell R.H. Curr. Biol. 2002; 12: 885-893Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). One of the major defects in fab1Δ cells, which are unable to make PtdIns(3,5)P2, is the failure to sort some proteins into multivesicular bodies (MVBs) (7Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 8Dove S.K. McEwen R.K. Mayes A. Hughes D.C. Beggs J.D. Michell R.H. Curr. Biol. 2002; 12: 885-893Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), but the molecular basis for this defect is poorly understood. A mammalian orthologue of Fab1p, named PIKfyve, was originally identified in insulin-sensitive tissues using a differential display screen (9Shisheva A. Sbrissa D. Ikonomov O. Mol. Cell. Biol. 1999; 19: 623-634Crossref PubMed Scopus (101) Google Scholar). PIKfyve is largely localized to late endosomal membranes (10Shisheva A. Rusin B. Ikonomov O.C. DeMarco C. Sbrissa D. J. Biol. Chem. 2001; 276: 11859-11869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) where the local synthesis of PtdIns(3,5)P2 may be regulated by insulin action in some cell types (11Sbrissa D. Ikonomov O. Shisheva A. Mol. Cell. Endocrinol. 2001; 181: 35-46Crossref PubMed Scopus (22) Google Scholar, 12Shisheva A. Cell Biol. Int. 2001; 25: 1201-1206Crossref PubMed Scopus (70) Google Scholar). Expression of a kinase-dead mutant of PIKfyve in mammalian cells results in the dilation and vacuolation of endosomal membranes, drastically altering cell morphology (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). This phenotype is consistent with the fab1Δ phenotype seen in yeast, suggesting a conserved role for PtdIns(3,5)P2 in MVB formation throughout the evolution of eukaryotic organisms. The sorting of membrane proteins into MVBs, often leading to their hydrolysis in lysosomal compartments is an important process in regulating their cellular levels. Degradation of membrane proteins may have particular importance in the control of cell signaling, by mediating the down-regulation of activated receptors such as the EGF receptor (14Piper R.C. Luzio J.P. Traffic. 2001; 2: 612-621Crossref PubMed Scopus (163) Google Scholar).Most phosphoinositide species have known effector molecules with modular phosphoinositide binding domains such as pleckstrin homology (PH), FYVE, or PX domains (15Cullen P.J. Cozier G.E. Banting G. Mellor H. Curr. Biol. 2001; 11: 882-893Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). There are presently, however, only two reports of proteins binding specifically to PtdIns(3,5)P2: centaurin-β2, which has a PH domain with binding specificity for this phosphoinositide (16Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Biochem. J. 2000; 351: 19-31Crossref PubMed Scopus (473) Google Scholar) and sorting nexin 1, which localizes exclusively to early or sorting endosomes (17Cozier G.E. Carlton J. McGregor A.H. Glesson P.A. Teasdale R.D. Mellor H. Cullen P.J. J. Biol. Chem. 2002; 277: 48730-48736Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The aim of this study has been to identify PtdIns(3,5)P2-binding proteins in rat adipocytes. These have elevated levels of PIKfyve compared with non-insulin-sensitive tissues and should therefore be a good source of PtdIns(3,5)P2 (9Shisheva A. Sbrissa D. Ikonomov O. Mol. Cell. Biol. 1999; 19: 623-634Crossref PubMed Scopus (101) Google Scholar, 12Shisheva A. Cell Biol. Int. 2001; 25: 1201-1206Crossref PubMed Scopus (70) Google Scholar).Previous methods for isolating novel phosphoinositide-binding proteins have been based on the affinity purification of proteins from cell/tissue extracts (18Tanaka K. Imajoh-Ohmi S. Sawada T. Shirai R. Hashimoto Y. Iwasaki S. Kaibuchi K. Kanaho Y. Shirai T. Terada Y. Kimura K. Nagata S. Fukui Y. Eur. J Biochem. 1997; 245: 512-519Crossref PubMed Scopus (82) Google Scholar, 19Krugmann S. Anderson K.E. Ridley S.H. Risso N. McGregor A. Coadwell J. Davidson K. Eguinoa A. Ellson C.D. Lipp P. Manifava M. Ktistakis N. Painter G. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Dove S.K. Michell R.H. Grewal A. Nazarian A. Erdjument-Bromage H. Tempst P. Stephens L.R. Hawkins P.T. Mol. Cell. 2002; 9: 95-108Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). However, a screen utilizing in vitro coupled transcription/translation system has recently been used for the same purpose, demonstrating the power of combining cDNA library technology with affinity isolation techniques (20Rao V.R. Corradetti M.N. Chen J. Peng J. Yuan J. Prestwich G.D. Brugge J.S. J. Biol. Chem. 1999; 274: 37893-37900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The screen described here also combines affinity purification with cDNA libraries but is based on phage display technology. This technology has been used in the selection of proteins able to bind to target ligands such as IgE (21Crameri R. Jaussi R. Menz G. Blaser K. Eur. J Biochem. 1994; 226: 53-58Crossref PubMed Scopus (146) Google Scholar, 22Lindborg M. Magnusson C.G. Zargari A. Schmidt M. Scheynius A. Crameri R. Whitley P. J. Invest. Dermatol. 1999; 113: 156-161Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), RNA (23Danner S. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12954-12959Crossref PubMed Scopus (115) Google Scholar), and small natural product molecules (24Sche P.P. McKenzie K.M. White J.D. Austin D.J. Chem. Biol. 1999; 6: 707-716Abstract Full Text PDF PubMed Scopus (141) Google Scholar). These targets are not readily amenable to the two-hybrid technologies commonly used in the identification of interactions between two proteins.We report the use of a phage display screen in the isolation of PtdIns(3,5)P2-binding proteins from a complex library containing a vast excess of nonspecific proteins. In addition we report the characterization of one of the selected proteins, mVps24p, and demonstrate that it is selective in its binding to PtdIns(3,5)P2. Furthermore, we show that the subcellular localization of mVps24p and cellular phenotype associated with expression of mVps24p derivatives is consistent with it being a PtdIns(3,5)P2 effector in vivo.EXPERIMENTAL PROCEDURESConstruction of Adipocyte Phage Display Library—Epididymal fat pads were removed from male Wistar rats and directly frozen in liquid nitrogen. Total RNA was extracted from the tissues using TRIzol (Invitrogen, UK) following the manufacturer's instructions. Polyadenylated mRNA was purified using the PolyATract mRNA isolation system (Promega, UK) and quantified by measuring absorbance at 260 nm. Randomly primed cDNA was synthesized from 4 μg of adipocyte mRNA and directionally cloned into the T7Select10-3 vector arms using the T7Select10-3 OrientExpress cDNA cloning system (Novagen). The cloned cDNA was packaged into phage particles, and a serial dilution was made of a small proportion (5%) of the packaged phage to determine the primary size of the library. To make an amplified phage lysate for biopanning, the remainder of the phage were used to infect and lyse Escherichia coli strain BLT5615. All the packaging, titration, and preparation of phage lysates were performed according to protocols provided with the T7Select10-3 cloning system.Synthesis of Biotinylated PtdIns(3,5)P 2 —The protected 3,5-phosphoinositide precursor (1, Fig. 1) was synthesized as previously described (25Riley A.M. Potter B.V.L. Tetrahedron Lett. 1998; 39: 6769-6772Crossref Scopus (24) Google Scholar). Reaction of 1 with phosphoramidite 2 (25Riley A.M. Potter B.V.L. Tetrahedron Lett. 1998; 39: 6769-6772Crossref Scopus (24) Google Scholar) followed by oxidation with m-chloroperoxybenzoic acid gave 3, which was then subjected to hydrogenolysis over palladium on carbon to provide the sn-2-aminohexanoyl PtdIns(3,5)P2 derivative 4. Reaction of the primary amine group of 4 with N-biotinylaminohexanoyl succinimide ester 5 in dimethylformamide/triethylammonium bicarbonate buffer (pH 8.2) gave biotinylated PtdIns(3,5)P2 6 in a manner previously described (20Rao V.R. Corradetti M.N. Chen J. Peng J. Yuan J. Prestwich G.D. Brugge J.S. J. Biol. Chem. 1999; 274: 37893-37900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) for the synthesis of biotinylated PtdIns(3,4,5)P3. Products were purified chromatographically. Other phosphoinositides and inositol phosphates used in this study were purchased from Echelon Research Laboratories Inc. and Alexis Biochemicals, respectively.Biopanning against Biotinylated PtdIns(3,5)P 2 —A slurry of immobilized NeutrAvidin (500 μl) (Pierce) was washed three times with 1 ml of binding buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 5 mm dithiothreitol) and resuspended in 1 ml of the same buffer. Biotinylated PtdIns(3,5)P2 (50 μlofa500 μm solution) was added to the beads, and the mixture was incubated for 1 h at 4 °C. The beads were washed three times with 1 ml of binding buffer and resuspended in 250 μl of the same buffer. 50 μl of these NeutrAvidin PtdIns(3,5)P2 beads were placed into siliconized 1.5-ml microcentrifuge tubes diluted in 1 ml of binding buffer. 100 μl of amplified phage lysate (∼1010 plaque forming units) was added to the tube and incubated at 18 °C with rotation for 1 h. The beads were then washed 12 times with 1 ml of binding buffer to remove unbound phage. Bound phage were eluted in 500 μl of binding buffer containing 1% SDS. A small aliquot (10 μl) of the eluted phage was serially diluted and used in a titration assay to determine the number of phage eluted. To make an enriched phage lysate, the remainder was used to infect and lyse BLT5615. The enriched phage lysate was used in the next round of biopanning, and the process was repeated three times before the eluted phage were plated, and individual plaques were picked. The cDNA inserts were amplified using PCR and sequenced following protocols supplied with the T7Select cloning system.DNA Manipulations and Expression—Standard protocols for recombinant DNA manipulations were used. The coding region of rat VPS24 was amplified from phage containing the VPS24 cDNA insert using PCR and oligonucleotides containing convenient restriction sites for cloning into expression vectors. For expression in E. coli VPS24 was subcloned into pET15b (Novagen). His6-mVps24p was expressed in E. coli strain BL21(DE3)pLysS and purified using Talon (Clontech) metal affinity resin using standard methods. Purified protein was dialyzed extensively against 50 mm Hepes, pH 7.2, 100 mm NaCl, 0.5 mm EDTA and stored at 4 °C. For expression in mammalian tissue culture cells VPS24 was subcloned into pFLAG-CMV5c (Sigma).Rat VPS4 cDNA was cloned by PCR amplification using oligonucleotide primers designed against the mouse VPS4 cDNA sequence. A Marathon (Clontech) cDNA library constructed according to the manufacturer's instructions from mRNA isolated from rat adipocytes was used as the template. The amplified PCR product was purified and ligated into the blunt cloning vector pT7-Blue3 (Novagen), and the correct sequence was confirmed by DNA sequencing. The QuikChange mutagenesis protocol (Stratagene) was used to make the Vps4 E235Q mutation. The coding region of Vps4 WT and Vps4 E235Q was subcloned into the mammalian expression vector pEGFPC1 (Clontech) to express these proteins with enhanced green fluorescent protein GFP at their N termini.Antibodies—An anti-mVps24 antibody was raised in rabbits, with His6-mVps24p as an antigen. The antibody was affinity-purified against the same antigen covalently coupled to Reacti-Gel (6×) (Pierce). The rabbit anti-rat ciM6PR was as described previously (26Reaves B.J. Row P.E. Bright N.A. Luzio J.P. Davidson H.W. J. Cell Sci. 2000; 113: 4099-4108PubMed Google Scholar) and was a gift of Dr. J. P. Luzio, University of Cambridge; the mouse anti-FLAG was purchased from Sigma; and the AlexaFluor 488 goat anti-mouse IgG, AlexaFluor 546 goat anti-rabbit IgG, and AlexaFluor 633 goat anti-mouse IgG were purchased from Molecular Probes (Eugene, OR).Binding of mVps24p to PtdIns(3,5)P 2 Beads and Phosphoinositide Competition for mVps24p Binding to PtdIns(3,5)P 2 Beads—Biotinylated PtdIns(3,5)P2 (typically 1 nmol for phosphoinositide competition experiments or amount indicated in individual experiments) was incubated with 25 μl of NeutrAvidin beads that had been pre-equilibrated in binding buffer (50 mm Hepes, pH 7.2, 100 mm NaCl, 0.5 mm EDTA) in a total volume of 500 μl for 30 min. The beads were washed twice with 1 ml of binding buffer and then resuspended in 500 μl of the same buffer. Recombinant His6-mVps24p (5 μg) was added to the NeutrAvidin PtdIns(3,5)P2 beads and incubated at 18 °C for 1 h with rotation. The beads were washed three times with 1 ml of detergent buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 5 mm dithiothreitol), and the final pellet was resuspended in SDS-PAGE sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol). Samples were analyzed for mVps24p by 12% SDS-PAGE and immunoblotting. The bead competition assays followed the same format described above except that the His6-mVps24p was mixed with competing phosphoinositides (typically 4 nmol of competitor was used) prior to incubation with the NeutrAvidin-PtdIns(3,5)P2 beads.Liposome Binding—Phosphatidylethanolamine/phosphatidylcholine liposomes containing the amounts of phosphoinositide indicated in the legends to Figs. 3, 4, and 5 were prepared as described by Patki et al. (27Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar). Liposomes (100 μg of total lipid) in 100 μl of binding buffer (50 mm Hepes, pH 7.2, 100 mm NaCl, 0.5 mm EDTA) were incubated with 5 μg of His6-mVps24p at 18 °C for 30 min. Samples were cooled on ice and centrifuged at 20,000 × g for 15 min at 4 °C. The supernatants were removed, and SDS-PAGE sample buffer (30 μl) was added to the pellets. Samples were analyzed for His6-mVps24p by 12% SDS-PAGE and immunoblotting.Fig. 3Adsorption of mVps24p by immobilized NeutrAvidin-PtdIns(3,5)P2. A, recombinant His6-mVps24p (5 μg) was incubated with NeutrAvidin beads having the indicated amounts of biotinylated-PtdIns(3,5)P2 coupled to their surface. The beads were washed to remove unbound His6-mVps24p. Recovery of His6-mVps24p bound to the beads was assessed by SDS-PAGE and immunoblotting with affinity purified anti-mVps24p antibody. B, cytosolic extracts (1 mg of protein) from rat adipocytes were incubated with NeutrAvidin beads (Neutravidin) or NeutrAvidin beads coupled to biotinylated-PtdIns(3,5)P2 (Neutravidin-PtdIns(3,5)P 2). The beads were washed to remove unbound proteins. Recovery of mVps24p bound to the beads was assessed by SDS-PAGE and immunoblotting with affinity purified anti-mVps24p antibody. Representative results from three separate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Analysis of the lipid binding specificity of His6-mVps24p. Analysis of interaction by competition assays. Recombinant His6-mVps24p (5 μg) was mixed with the indicated competitor phosphoinositide (4 nmol) and then incubated with NeutrAvidin-PtdIns(3,5)P2 beads (1 nmol of biotinylated-PtdIns(3,5)P2) as described under “Experimental Procedures.” Following incubation, the beads were washed to remove unbound His6-mVps24p. Recovery of His6-mVps24p bound to the beads was assessed by SDS-PAGE and immunoblotting with affinity-purified anti-mVps24p antibody. A, competition with C8 phosphoinositides and inositol phosphate head groups. B, competition with C16 phosphoinositides. Representative results from three and six experiments are shown for A and B, respectively. Analysis of binding of mVps24p to liposomes. Recombinant His6-mVps24p (5 μg) was incubated with liposomes (100 μg) consisting of PE/PC and the indicated phosphoinositide, prepared as described under “Experimental Procedures.” The liposomes were pelleted by centrifugation and recovery of His6-mVps24p bound to the liposomes was assessed by SDS-PAGE and immunoblotting with affinity purified anti-mVps24p antibody. C, liposomes reconstituted with 5 mol % of the indicated phosphoinositide or phosphatidylserine (PS). D, liposomes reconstituted with 0, 1, 2, 3, 4, and 5 mol % of PtdIns(3,5)P2. Representative results from three and two experiments are shown for C and D, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Mutation of a conserved positively charged amino acid residue reduces PtdIns(3,5)P2 binding. A, sequence alignment of rat/mouse CHMPs. Sequences were aligned using ClustalX (default parameters), and amino acids conserved in greater than 50% of sequences were shaded using BOXSHADE. GenBank™ accession numbers for the mouse and rat proteins are: CHMP1 (mouse), NP_663581; CHMP2 (mouse), NP_081161; CHMP3 (rat, this study), AY150169; CHMP4 (rat), XP_231625; CHMP5 (mouse), NP_084090; and CHMP6 (mouse), BB610671. The conserved positively charged amino acid that was mutated in mVps24p/CHMP3 is marked with an asterisk. B, recombinant His6-mVps24p (WT) or mutant His6-mVps24p (K49D) (1 μg) was incubated with liposomes (100 μg) consisting of PE/PC or PE/PC spiked with 2 mol% of PtdIns(3,5)P2, prepared as described under “Experimental Procedures.” The liposomes were pelleted by centrifugation. His6-mVps24p bound to the liposomes pellet (P) and that remaining in the supernatant (S) was assessed by SDS-PAGE and immunoblotting with affinity purified anti-mVps24p antibody. Representative results from three separate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Affinity Isolation of mVps24p from Rat Adipocyte Cytosol—NeutrAvidin-PtdIns(3,5)P2 beads were prepared as described previously for the PtdIns(3,5)P2-bead binding experiments except that 2.5 nmol of biotinylated PtdIns(3,5)P2 was bound to each 25 μl of beads. Control beads were mock treated without the addition of biotinylated PtdIns(3,5)P2. For each experiment, 25 μl of beads was incubated with 1 mg of cytosol from rat adipocytes in binding buffer for 1 h at 18 °C. The beads were washed five times with 1 ml of the binding buffer, and after the final wash the beads were resuspended in 30 μl of SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE and by immunoblotting for the presence of mVps24p using anti-mVps24 affinity-purified polyclonal antibodies.Cell Culture, Transfection, and Immunofluorescence Microscopy—COS-7 cells were grown in Dulbecco's modified minimal essential medium (Invitrogen) containing 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were plated onto glass coverslips and grown until ∼60% confluent prior to transfection using GeneJuice transfection reagent (Novagen) according to manufacturer's instructions. Cells grown on glass coverslips were transfected 18-24 h prior to fixation and processing for immunofluorescence microscopy. Cells were rinsed with PBS and fixed in 2% paraformaldehyde in PBS for 15 min prior to permeabilizing with 0.1% Triton X-100 for 5 min at room temperature. Nonspecific binding sites were blocked with 10% fetal calf serum for 20 min prior to addition of primary antibodies. Double labeling of Vps24p and M6PR was carried out using mouse anti-FLAG (10 μg/ml final) and rabbit anti-rat ciM6PR followed by washing and addition of Alexa 488 goat anti-mouse IgG and Alexa 546 goat anti-rabbit IgG at 5-8 μg/ml in 10% fetal calf serum-PBS. Coverslips were washed and mounted in Mowiol (Calbiochem, San Diego, CA). In cells expressing GFP-Vps4E235Q, rabbit anti-M6PR was detected using Alexa 546 goat anti-rabbit IgG and Vps24-FLAG using Alexa 633 goat anti-mouse IgG. The labeled cells were viewed, and images were obtained on a Zeiss LSM510 laser scanning confocal microscope or on a Nikon Eclipse fluorescent microscope.RESULTSIdentification of PtdIns(3,5)P2 Binding Proteins Using Phage Display—The affinity ligand (biotin-PtdIns(3,5)P2) (6, Fig. 1) was immobilized on NeutrAvidin beads. A T7Select10-3 adipocyte library was constructed that contained 2 × 106 independent phage. Phage selected at random from the un-amplified library all contained cDNA inserts over 200 base pairs in length.Following the first round of biopanning against the NeutrAvidin PtdIns(3,5)P2 beads, the titer of the eluted phage was 2 × 105. The eluted titer was 1.5 × 106 in the second round and 5 × 106 in the third, a 25-fold overall increase. This was an indicator of successful biopanning, because the enrichment of phage that bind the target molecule during early rounds usually results in increases in titer for recovered phage in later rounds (T7-Select manual, Novagen). Following the third round of biopanning, individual plaques were picked, and their cDNA inserts were amplified by PCR and then sequenced. Sequencing of 12 plaques revealed only two different cDNA inserts, indicating a highly specific enrichment from the initial library, potentially containing 2 × 106 different clones. Only one of the cDNA inserts is considered here, encoding for an open reading frame (ORF) corresponding to 230 amino acids, in-frame with the T7-capsid protein (Fig. 2A). The ORF shared a high degree of sequence identity (30%) with Saccharomyces cerevisiae Vps24p (Fig. 2B). S. cerevisiae Vps24p is a vacuolar protein-sorting protein of the class-E type (28Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (671) Google Scholar, 29Babst M. Wendland B. Estepa E.J. Emr S.D. EMBO J. 1998; 17: 2982-2993Crossref PubMed Scopus (613) Google Scholar). We therefore named the protein mammalian Vps24 protein (mVps24p). The sequence of mVps24p lacks any previously identified phosphoinositide binding domains. It is predicted to be a 223-amino acid (7Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar amino acids in the 230-amino acid ORF were upstream of the start methionine), 25-kDa hydrophilic protein (pI = 5) that has no long hydrophobic stretches of amino acids. One noticeable feature is that the charge distribution of the protein is extremely biased, with the N-terminal half being very basic (pI = 10.2) and the C-terminal half being acidic (pI = 3.9).Fig. 2Sequence analysis of PtdIns(3,5)P2 binding phage. A, nucleotide and deduced amino acid sequence of the cDNA insert in a pT7 phage selected by its ability to bind PtdIns(3,5)P2 beads. The first three amino acids are derived from the phage capsid protein. The predicted start codon and first methionine are shown in boldface type. Amino acids in the ORF that are upstream of the start codon and that are predicted not to be present in the natural protein are underlined. B, alignment of the deduced amino acid sequence (Rat) and S. cerevisiae Vps24p (S.cer). Identical amino acids are shaded in black; conserved amino acids are gray.View Large" @default.
• W2023198417 created "2016-06-24" @default.
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• W2023198417 date "2003-10-01" @default.
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• W2023198417 title "Identification of Mammalian Vps24p as an Effector of Phosphatidylinositol 3,5-Bisphosphate-dependent Endosome Compartmentalization" @default.
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