Vacuole Membrane Topography of the DPP1-encoded Diacylglycerol Pyrophosphate Phosphatase Catalytic Site from Saccharomyces cerevisiae
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m311779200
ISSN1083-351X
AutoresGil‐Soo Han, Celeste N. Johnston, George Carman,
Tópico(s)Autophagy in Disease and Therapy
ResumoThe Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the β-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane. The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the β-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane. DGPP 1The abbreviations used are: DGPPdiacylglycerol pyrophosphatePAphosphatidatePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineHAhemagglutininAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidMPBNα-(3-maleimidylpropionyl) biocytin. phosphatase, encoded by the DPP1 gene (1Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 2Faulkner A.J. Chen X. Rush J. Horazdovsky B. Waechter C.J. Carman G.M. Sternweis P.C. J. Biol. Chem. 1999; 274: 14831-14837Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 3Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar), is a 34-kDa vacuole membrane-associated enzyme (4Han G.-S. Johnston C.N. Chen X. Athenstaedt K. Daum G. Carman G.M. J. Biol. Chem. 2001; 276: 10126-10133Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) that was first discovered in the yeast Saccharomyces cerevisiae (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It catalyzes the removal of the β-phosphate from DGPP to form PA, and it then removes the phosphate from PA to form diacylglycerol (Fig. 1) (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). DGPP is the preferred substrate, and PA will not serve as a substrate in the presence of DGPP (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). DGPP phosphatase also utilizes lyso-PA (6Dillon D.A. Wu W.-I. Riedel B. Wissing J.B. Dowhan W. Carman G.M. J. Biol. Chem. 1996; 271: 30548-30553Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), sphingoid base phosphates (7Dillon D.A. Chen X. Zeimetz G.M. Wu W.-I. Waggoner D.W. Dewald J. Brindley D.N. Carman G.M. J. Biol. Chem. 1997; 272: 10361-10366Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and isoprenoid phosphates (2Faulkner A.J. Chen X. Rush J. Horazdovsky B. Waechter C.J. Carman G.M. Sternweis P.C. J. Biol. Chem. 1999; 274: 14831-14837Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) as substrates in vitro. However, only DGPP and PA have been shown to be substrates in vivo (8Toke D.A. Bennett W.L. Oshiro J. Wu W.I. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 273: 14331-14338Abstract Full Text Full Text PDF Scopus (104) Google Scholar). In fact, the products of the LCB3/LBP1/YSR2 (9Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 16110-16117Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Mandala S.M. Thornton R. Tu Z.X. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 11Mao C.G. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and the CWH8 (12Fernandez F. Rush J.S. Toke D.A. Han G.S. Quinn J.E. Carman G.M. Choi J.Y. Voelker D.R. Aebi M. Waechter C.J. J. Biol. Chem. 2001; 276: 41455-41464Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) genes are responsible for the dephosphorylation of sphingoid base phosphates and isoprenoid phosphates in vivo, respectively. diacylglycerol pyrophosphate phosphatidate phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine hemagglutinin 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid Nα-(3-maleimidylpropionyl) biocytin. The DGPP molecule was originally identified in plants as the product of the PA kinase enzyme (13Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Crossref PubMed Scopus (46) Google Scholar). Research with plants indicates that DGPP may function as a signaling molecule under stress conditions. It accumulates upon G-protein activation (14Munnik T. de Vrije T. Irvine R.F. Musgrave A. J. Biol. Chem. 1996; 271: 15708-15715Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), hyperosmotic stress (15Munnik T. Meijer H.J.G. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant J. 2000; 22: 147-154Crossref PubMed Google Scholar), dehydration (15Munnik T. Meijer H.J.G. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant J. 2000; 22: 147-154Crossref PubMed Google Scholar), Rhizobium-secreted nodulation factors (16Den Hartog M. Musgrave A. Munnik T. Plant J. 2001; 25: 55-65Crossref PubMed Google Scholar), and general elicitors such as xylanase (17Van der Luit A.H. Piatti T. Van Doorn A. Musgrave A. Felix G. Boller T. Munnik T. Plant Physiol. 2000; 123: 1507-1515Crossref PubMed Scopus (187) Google Scholar). DGPP accumulation is transient and coincides with a rise in PA levels (15Munnik T. Meijer H.J.G. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant J. 2000; 22: 147-154Crossref PubMed Google Scholar, 17Van der Luit A.H. Piatti T. Van Doorn A. Musgrave A. Felix G. Boller T. Munnik T. Plant Physiol. 2000; 123: 1507-1515Crossref PubMed Scopus (187) Google Scholar). Because of the reactions catalyzed by DGPP phosphatase, the enzyme may play a role during stress conditions to regulate specific cellular pools of DGPP and PA (18Oshiro J. Han G.-S. Carman G.M. Biochim. Biophys. Acta. 2003; 1635: 1-9Crossref PubMed Scopus (27) Google Scholar). Indeed, the Arabidopsis thaliana homolog (AtLPP1) of the yeast DPP1 gene is induced by genotoxic stress (γ and UV-B radiation), G-protein activation, and oxidative stress (19Pierrugues O. Brutesco C. Oshiro J. Gouy M. Deveaux Y. Carman G.M. Thuriaux P. Kazmaier M. J. Biol. Chem. 2001; 276: 20300-20308Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Consistent with these observations, the S. cerevisiae DPP1-encoded DGPP phosphatase is induced by the stress conditions of zinc depletion (4Han G.-S. Johnston C.N. Chen X. Athenstaedt K. Daum G. Carman G.M. J. Biol. Chem. 2001; 276: 10126-10133Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and stationary phase (20Oshiro J. Rangaswamy S. Chen X. Han G.-S. Quinn J.E. Carman G.M. J. Biol. Chem. 2000; 275: 40887-40896Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and by inositol supplementation (20Oshiro J. Rangaswamy S. Chen X. Han G.-S. Quinn J.E. Carman G.M. J. Biol. Chem. 2000; 275: 40887-40896Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Moreover, the expression of the DPP1 homolog in the pathogenic yeast Candida albicans is induced in clinical isolates that are resistant to azole antifungal agents (21Rogers P.D. Barker K.S. Antimicrob. Agents Chemother. 2003; 47: 1220-1227Crossref PubMed Scopus (130) Google Scholar). The catalytic activity of DGPP phosphatase is conferred by a three-domain lipid phosphatase motif (22Stukey J. Carman G.M. Protein Sci. 1997; 6: 469-472Crossref PubMed Scopus (222) Google Scholar) found in the lipid phosphatase superfamily (23Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 24Brindley D.N. English D. Pilquil C. Buri K. Ling Z.C. Biochim. Biophys. Acta. 2002; 1582: 33-44Crossref PubMed Scopus (104) Google Scholar). The conserved Arg125, His169, and His223 residues within domains I, II, and III, respectively, are required for both the DGPP phosphatase and PA phosphatase activities of the enzyme (25Toke D.A. McClintick M.L. Carman G.M. Biochemistry. 1999; 38: 14606-14613Crossref PubMed Scopus (39) Google Scholar). The lipid phosphatase activities of DGPP phosphatase are Mg2+-independent and N-ethylmaleimide-insensitive (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The PA phosphatase activity of the DGPP phosphatase is distinct (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) from the Mg2+dependent and N-ethylmaleimide-sensitive PA phosphatase enzyme (26Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Abstract Full Text PDF PubMed Google Scholar, 27Morlock K.R. McLaughlin J.J. Lin Y.-P. Carman G.M. J. Biol. Chem. 1991; 266: 3586-3593Abstract Full Text PDF PubMed Google Scholar) that is presumably responsible for the synthesis of phospholipids and triacylglycerols in S. cerevisiae (28Carman G.M. Biochim. Biophys. Acta. 1997; 1348: 45-55Crossref PubMed Scopus (70) Google Scholar). In addition to yeast, DGPP phosphatase activity is found in a wide range of organisms (e.g. plants, bacteria, and mammalian cells (19Pierrugues O. Brutesco C. Oshiro J. Gouy M. Deveaux Y. Carman G.M. Thuriaux P. Kazmaier M. J. Biol. Chem. 2001; 276: 20300-20308Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 29Riedel B. Morr M. Wu W.-I. Carman G.M. Wissing J.B. Plant Sci. 1997; 128: 1-10Crossref Scopus (14) Google Scholar)), suggesting an important role for the enzyme in cell physiology (28Carman G.M. Biochim. Biophys. Acta. 1997; 1348: 45-55Crossref PubMed Scopus (70) Google Scholar). Deletion of the DPP1 gene results in altered phospholipid composition (8Toke D.A. Bennett W.L. Oshiro J. Wu W.I. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 273: 14331-14338Abstract Full Text Full Text PDF Scopus (104) Google Scholar). dpp1Δ mutant cells exhibit increased amounts of DGPP and PA and a decrease in the amount of PI (8Toke D.A. Bennett W.L. Oshiro J. Wu W.I. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 273: 14331-14338Abstract Full Text Full Text PDF Scopus (104) Google Scholar). Moreover, DPP1 is one of the most highly regulated genes that respond to zinc depletion in the S. cerevisiae genome (3Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar, 30Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). However, the physiological role of the DGPP phosphatase enzyme is not fully understood because dpp1Δ mutant cells do not exhibit dramatic phenotypic changes under standard laboratory conditions (1Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 2Faulkner A.J. Chen X. Rush J. Horazdovsky B. Waechter C.J. Carman G.M. Sternweis P.C. J. Biol. Chem. 1999; 274: 14831-14837Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 3Yuan D.S. Genetics. 2000; 156: 45-58Crossref PubMed Google Scholar). The subcellular location of DGPP phosphatase to the vacuole membrane suggests that the functional role of the enzyme utilizing phospholipid substrates should be governed by the topography of its catalytic site. In this report, we provided evidence based on protease protection analysis, chemical labeling, and enzyme activity measurements that the DGPP phosphatase catalytic site was oriented to the cytosolic face of the vacuole membrane. In addition, we demonstrated that the enzyme substrates DGPP and PA were present in the vacuole membrane and that their levels were significantly reduced when cells were depleted for zinc. Materials—All of the chemicals were reagent grade. Growth medium supplies were purchased from Difco Laboratories. Protease inhibitors (aprotinin, benzamidine, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride), Ficoll 400, lyticase, 4-(2-pyridylazo)resorcinol, bovine serum albumin, urea, Triton X-100, and endoproteinase Lys-C were purchased from Sigma. Protein assay reagent, electrophoresis reagents, and molecular mass markers were purchased from Bio-Rad. Mouse monoclonal anti-HA antibodies (12CA5) and agarose-conjugated anti-HA antibodies were purchased from Roche Applied Science. FM4–64, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS), Nα-(3-maleimidylpropionyl)biocytin (MPB), monoclonal antibodies against yeast carboxypeptidase Y, and vacuolar H+-ATPase were purchased from Molecular Probes. Alkaline phosphatase-linked goat anti-rabbit (or mouse) IgG antibodies were purchased from Pierce. Radiochemicals were from PerkinElmer Life Sciences. Scintillation counting supplies were from National Diagnostics. Liqui-Nox was from Alconox, Inc. Polyvinylidene difluoride membranes and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. Silica Gel 60 high performance TLC plates were from EM Science, and phospholipids were obtained from Avanti Polar Lipids. Strains and Growth Conditions—S. cerevisiae strain W303–1A (MATaade2–1 can1–100 his3–11, 15 leu2–3, 112 trp1–1 ura3–1) (31Thomas B. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1355) Google Scholar) is the parent of the dpp1Δ mutant strain DTY1 (MATaade2–1 can1–100 his3–11, 15 leu2–3, 112 trp1–1 ura3–1 dpp1Δ::TRP1/Kanr) (1Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The HA-tagged version of the DPP1 gene was expressed in DTY1 from plasmid pGH202 (4Han G.-S. Johnston C.N. Chen X. Athenstaedt K. Daum G. Carman G.M. J. Biol. Chem. 2001; 276: 10126-10133Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), which is a derivative of YEp351 (32Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google Scholar). Cultures were grown at 30 °C in YPDA medium (1% yeast extract, 2% peptone, 2% glucose, and 25 mg/liter adenine) or in synthetic complete medium (33Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar) containing 2% glucose. The appropriate amino acid of synthetic complete medium was omitted for selection purposes. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. Cultures were grown to the exponential phase (1–2 × 107 cells/ml) and harvested by centrifugation at 1,500 × g for 5 min. For zinc-depleted cultures, cells were first grown for 24 h in synthetic complete medium supplemented with 1.4 μm zinc sulfate. Cultures were harvested, washed in de-ionized distilled water, diluted to 1 × 106 cells/ml in media containing 0 or 1.4 μm zinc sulfate, and grown for 24 h. Cultures were then diluted to 1 × 106 cells/ml and grown in media containing 0 or 1.4 μm zinc sulfate. This process was used to deplete internal stores of zinc. For zinc-free glassware, items were washed with Liqui-Nox and rinsed several times with distilled water followed by a final rinse with de-ionized distilled water. Preparation of Cell Extracts and Vacuoles—Cell extracts were prepared as described previously (34Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar, 35Oshiro J. Han G.-S. Iwanyshyn W.M. Conover K. Carman G.M. J. Biol. Chem. 2003; 278: 31495-31503Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Cells were suspended in 50 mm Tris maleate buffer (pH 7.0) containing 1 mm EDTA, 0.3 m sucrose, 10 mm 2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Cells were disrupted by homogenization with chilled glass beads (0.5-mm diameter) using a Biospec Products bead beater. Samples were homogenized for 10 1-min bursts followed by a 2-min cooling between bursts at 4 °C. The cell extract (supernatant) was obtained by centrifugation of the homogenate at 1,500 × g for 10 min. Vacuoles were isolated from spheroplasts by several steps of flotation and density gradient centrifugation as described by Uchida et al. (36Uchida E. Ohsumi Y. Anraku Y. Methods Enzymol. 1988; 157: 544-562Crossref PubMed Scopus (34) Google Scholar). The protein concentration of the cell extract and vacuoles was determined by the method of Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217547) Google Scholar) using bovine serum albumin as the standard. Staining of Vacuoles with FM4–64—Vacuolar membranes were stained with the specific styryl dye FM4–64 (38Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1141) Google Scholar) at a final concentration of 50 μm. Fluorescent images were observed and recorded using an Olympus BH2-RFCA fluorescence microscope equipped with a Photometrics Sensys KAF-1400 CCD camera. SDS-PAGE and Immunoblot Analysis—Proteins were separated by SDS-PAGE (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) using 10% gels or 10–20% gradient gels. Proteins were transferred to polyvinylidene difluoride membranes for immunoblotting as described by Haid and Suissa (40Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Crossref PubMed Scopus (233) Google Scholar). Membranes were probed with an appropriate dilution of the indicated primary antibody. Goat anti-rabbit or anti-mouse IgG alkaline phosphatase conjugate was used as a secondary antibody against primary antibodies produced in rabbits or mice, respectively, at a dilution of 1:5000. Antibody interactions were detected using the enhanced chemifluorescence Western blotting detection kit as described by the manufacturer. Signals on the membrane were acquired by fluoroimaging analysis. The relative density of the protein signals was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability. Endoproteinase Lys-C Treatment of Vacuoles—Vacuoles (5 μg of protein) were mixed with 1 μg of endoproteinase Lys-C and incubated for 2 h at 37 °C in a total volume of 15 μl. The reaction mixture contained 50 mm Tris-HCl (pH 8.5), 0.3 m sucrose, and 3 m urea. Urea was included in the reaction mixture, because it facilitated exposure of lysine residues to endoproteinase Lys-C and provided some protection from the endogenous vacuolar protease(s) that digested the DGPP phosphatase in the detergent-solubilized vacuole. Vacuoles were ruptured by including 0.4% Triton X-100 in the reaction mixture. After incubation, 5 mm phenylmethylsulfonyl fluoride was added to the mixture to inhibit the protease. The reaction products were then subjected to SDS-PAGE with a 10–20% gradient gel followed by transfer to a polyvinylidene difluoride membrane. The membrane was probed with anti-HA antibodies or with antibodies raised against the C-terminal epitope (residues 263–279) of DGPP phosphatase (20Oshiro J. Rangaswamy S. Chen X. Han G.-S. Quinn J.E. Carman G.M. J. Biol. Chem. 2000; 275: 40887-40896Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Chemical Labeling of Cysteine Residues—The cysteine residues of vacuole membrane proteins were labeled with sulfhydryl reagents as described by Leng et al. (41Leng X.H. Nishi T. Forgac M. J. Biol. Chem. 1999; 274: 14655-14661Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) with some modifications. Vacuoles (15 μg of protein) were suspended in 200 μl of Tris-HCl buffer (pH 7.4) containing 0.3 m sucrose and incubated for 10 min at 25 °C in the absence or presence of 100 μm AMS, a membrane-impermeable reagent used to label cysteine residues. The reaction mixture was then diluted to 1 ml with vacuole suspension buffer and incubated for 20 min at 25 °C with 250 μm MPB, a membrane-permeable reagent used to label cysteine residues with biotin. The labeling reaction was terminated by the addition of 20 mm 2-mercaptoethanol. The sulfhydryl reagent-treated vacuoles were collected by centrifugation at 37,000 × g for 30 min at 4 °C. The vacuoles were lysed in 500 μl of radioimmunoprecipitation buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) (42Harlow E. Lane D. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) and mixed with 100 μl of 10% slurry of agarose-conjugated anti-HA antibodies for 2 h at 4 °C. The anti-HA affinity matrix was collected by centrifugation at 5,000 × g for 30 s, washed twice with radioimmunoprecipitation buffer, and resuspended in 20 μl of SDS-PAGE treatment buffer. After boiling and a brief centrifugation, the supernatant was subjected to SDS-PAGE with a 10–20% gradient gel followed by transfer to polyvinylidene difluoride membrane. The membrane was probed with alkaline phosphatase-conjugated NeutrAvidin to detect biotinylated proteins. The membrane was then stripped and re-probed with anti-HA antibodies to detect DGPP phosphatase. Preparation of Labeled DGPP Substrates—32P-Labeled DGPP was enzymatically synthesized from PA and ATP with purified Catharanthus roseus PA kinase as described by Wu et al. (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). [β-32P]DGPP was synthesized from PA and [γ-32P]ATP, whereas [α-32P]DGPP was synthesized from [32P]PA and ATP. The [32P]PA used in the latter reaction was synthesized from diacylglycerol and [γ-32P]ATP using Escherichia coli diacylglycerol kinase (43Walsh J.P. Bell R.M. J. Biol. Chem. 1986; 261: 6239-6247Abstract Full Text PDF PubMed Google Scholar). The 32P-labeled DGPP substrates were purified by thin-layer chromatography on potassium oxalate-treated plates using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4) (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). DGPP Phosphatase Assays—DGPP phosphatase activity was routinely measured by following the release of water-soluble 32Pi from chloroform-soluble [β-32P]DGPP (10,000 cpm/nmol) (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Unless otherwise indicated, the standard reaction mixture contained 50 mm citrate buffer (pH 5.0), 0.1 mm [β-32P]DGPP, 2 mm Triton X-100, and enzyme protein in a total volume of 0.1 ml. The function of Triton X-100 in the assay system is to solubilize the DGPP substrate (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). All of the enzyme assays were conducted in triplicate at 30 °C. The mean ± S.D. of the assays was ± 5%. DGPP phosphatase reactions were linear with time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of product/min. DGPP phosphatase activity was also measured with DGPP incorporated into phospholipid vesicles that were prepared by sonication (44Bangham A.D. Hesketh T.R. Kornberg H.L. Metcalfe J.C. Northcote D.H. Techniques in Lipid and Membrane Biochemistry, Part II. Elsevier Scientific Publishers, New York1982: 1-25Google Scholar). The phospholipid vesicles contained 0.18 mm PC, 0.125 mm PE, 0.125 mm PI, 0.062 mm PS, and 0.1 mm [α-32P]DGPP. The molar ratio of PC/PE/PI/PS in the vesicles was 3:2:2:1, the approximate composition of the major phospholipids in wild type S. cerevisiae (45Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). The molar ratio of the PC/PE/PI/PS mixture to DGPP was 5:1. In this assay, DGPP phosphatase activity was measured by following the formation of [32P]PA from [α-32P]DGPP (10,000 cpm/nmol) (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Following the reaction, phospholipid vesicles (supernatant) were separated from vacuoles by centrifugation at 37,000 × g for 30 min. Phospholipids were extracted (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar) and analyzed by thin-layer chromatography using chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4) as the solvent system (5Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The identity of the labeled phospholipids on the chromatography plates was confirmed by comparison with standard radioactive DGPP and PA. Radiolabeled DGPP and PA were visualized by phosphorimaging analysis, and their relative quantities were analyzed using ImageQuant software. Analysis of Vacuole Membrane Phospholipids—Phospholipids were extracted from vacuoles by the method of Bligh and Dyer (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). The aqueous phase and mat of solid material between the two phases of this extraction were removed with a Pasteur pipette and subjected to a second extraction with chloroform. The chloroform phases were combined, dried under nitrogen, and resuspended in 500 μl of chloroform/methanol (9:1, v/v). A 25-μl aliquot was used to determine the total phosphate content of the sample. The remainder was dried to a minimal volume under nitrogen. The sample was spotted onto an oxalate-EDTA (1.2% potassium oxalate, 2 mm EDTA dissolved in methanol/water (2:3, v/v)) treated high performance TLC Silica Gel 60 plate, which had been heated at 110 °C overnight and cooled to room temperature immediately prior to the application of the sample. The first dimension was developed using chloroform/methanol/ammonium hydroxide/water (30: 16.7:1.3:2, v/v). The plate was allowed to completely dry in the hood for at least 1 h. It was then dried in vacuo for an additional 2 h. The second dimension was developed using chloroform/methanol/acetic acid/water (32:4:5:1, v/v). The plate was allowed to dry completely overnight in the hood. Phospholipids were visualized by acid charring (47Kates M. Techniques of Lipidology. Isolation, Analysis, and Identification of Lipids. Elsevier, New
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