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Inactivation of the Phosphoinositide Phosphatases Sac1p and Inp54p Leads to Accumulation of Phosphatidylinositol 4,5-Bisphosphate on Vacuole Membranes and Vacuolar Fusion Defects

2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês

10.1074/jbc.m701038200

1083-351X

Fenny Wiradjaja, Lisa M. Ooms, Sabina Tahirović, Ellie Kuhne, Rodney J. Devenish, Alan L. Munn, Robert C. Piper, Peter Mayinger, Christina A. Mitchell,

Endoplasmic Reticulum Stress and Disease

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Δsac1 Δinp54 and sac1ts Δinp54 mutants. Furthermore, sac1ts Δinp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Δsac1 Δinp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects. Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Δsac1 Δinp54 and sac1ts Δinp54 mutants. Furthermore, sac1ts Δinp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Δsac1 Δinp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects. Phosphoinositide signaling molecules are phosphorylated derivatives of phosphatidylinositol (PtdIns) 3The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PH-OSBP, pleckstrin-homology domain of oxysterol binding protein; PH-PLC, pleckstrin-homology domain of phospholipase C; PH-Num1p, pleckstrin-homology domain of Num1p; CPY, carboxypeptidase Y; ALP, alkaline phosphatase; vps, vacuolar protein sorting; GFP, green fluorescent protein; ER, endoplasmic reticulum; HPLC, high pressure liquid chromatography; chr, chromosome; PH, pleckstrin homology; OSBP, oxysterol-binding protein; CMAC, 7-amino-4-chloromethylaminocoumarin; V-ATPase, vacuolar ATPase; OCRL, oculocerebrorenal protein of Lowe.3The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PH-OSBP, pleckstrin-homology domain of oxysterol binding protein; PH-PLC, pleckstrin-homology domain of phospholipase C; PH-Num1p, pleckstrin-homology domain of Num1p; CPY, carboxypeptidase Y; ALP, alkaline phosphatase; vps, vacuolar protein sorting; GFP, green fluorescent protein; ER, endoplasmic reticulum; HPLC, high pressure liquid chromatography; chr, chromosome; PH, pleckstrin homology; OSBP, oxysterol-binding protein; CMAC, 7-amino-4-chloromethylaminocoumarin; V-ATPase, vacuolar ATPase; OCRL, oculocerebrorenal protein of Lowe. that play critical roles regulating the actin cytoskeleton, cellular proliferation, and vesicular trafficking (1.Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (239) Google Scholar). PtdIns can be reversibly modified by lipid kinase phosphorylation of the D-3, D-4, or D-5 positions of the inositol head group to create phosphorylated phosphoinositides that recruit and activate effectors containing phosphoinositide-binding domains (1.Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (239) Google Scholar). In yeast and mammalian cells, phosphatidylinositol 4-phosphate (PtdIns(4)P) regulates secretion from the Golgi, and PtdIns(4)P recruitment of specific effector proteins, including FAPP1 and FAPP2, is required for mammalian Golgi to plasma membrane trafficking (1.Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (239) Google Scholar, 2.Hama H. Schnieders E.A. Thorner J. Takemoto J.Y. DeWald D.B. J. Biol. Chem. 1999; 274: 34294-34300Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3.Godi A. Di Campli A. Konstantakopoulos A. Di Tullio G. 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In yeast, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated from PtdIns(4)P by the PtdIns(4)P 5-kinase, Mss4p (8.Homma K. Terui S. Minemura M. Qadota H. Anraku Y. Kanaho Y. Ohya Y. J. Biol. Chem. 1998; 273: 15779-15786Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). PtdIns(4,5)P2 is involved in the regulation of endocytosis, actin cytoskeletal dynamics, and the maintenance of Golgi structural integrity (1.Roth M.G. Physiol. Rev. 2004; 84: 699-730Crossref PubMed Scopus (239) Google Scholar).Phosphorylated phosphoinositides are dephosphorylated by lipid phosphatases that regulate the temporal and spatial distribution of phosphoinositide signals. In yeast, PtdIns(4)P and PtdIns(4,5)P2 are hydrolyzed by phosphoinositide phosphatases, including Sac1p and the inositol polyphosphate 5-phosphatases (5-phosphatases), Inp51-4p (9.Wiradjaja F. Ooms L.M. Whisstock J.C. McColl B. Helfenbaum L. Sambrook J.F. Gething M.J. Mitchell C.A. J. Biol. Chem. 2001; 276: 7643-7653Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 10.Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 11.Stolz L.E. Kuo W.J. Longchamps J. Sekhon M.K. York J.D. J. Biol. Chem. 1998; 273: 11852-11861Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12.Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P. Gleeson P. Gething M.J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (31) Google Scholar). Sac1p is a polyphosphoinositide phosphatase containing a CX5R catalytic motif, which is found in both SacI domain-containing lipid phosphatases, as well as dual specificity tyrosine and serine/threonine phosphatases. Four active SacI domain-containing lipid phosphatases exist in S. cerevisiae, including Sac1p, Fig4p, and two of the four 5-phosphatases, Inp52p and Inp53p (also called Sjl2p and Sjl3p) (10.Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 13.Rudge S.A. Anderson D.M. Emr S.D. Mol. Biol. Cell. 2004; 15: 24-36Crossref PubMed Scopus (153) Google Scholar). The SacI domain of Inp51p/Sjl1p lacks the CX5R motif and is therefore catalytically inactive. Sac1p hydrolyzes PtdIns(4)P, PtdIns(3)P, and PtdIns(3,5)P2 to PtdIns, but PtdIns(4)P is the preferred substrate (10.Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Sac1p localizes to the endoplasmic reticulum (ER) by interaction with dolicholphosphate mannose synthase Dpm1p during the exponential phase of cell growth and translocates to the Golgi when nutrients become depleted (14.Faulhammer F. Konrad G. Brankatschk B. Tahirovic S. Knodler A. Mayinger P. J. Cell Biol. 2005; 168: 185-191Crossref PubMed Scopus (61) Google Scholar). Sac1p regulates Golgi membrane trafficking by controlling PtdIns(4)P levels (15.Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The sac1 null phenotype is complex, including altered phosphoinositide metabolism, accelerated phosphatidylcholine biosynthesis, cold sensitivity, inositol auxotrophy, hypersensitivity to multiple drugs, actin and cell wall defects, and delayed endocytic and vacuolar trafficking (16.Cleves A.E. Novick P.J. Bankaitis V.A. J. 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Cell. 1991; 64: 789-800Abstract Full Text PDF PubMed Scopus (284) Google Scholar).The 5-phosphatases Inp51-4p hydrolyze PtdIns(4,5)P2 forming PtdIns(4)P via their central 5-phosphatase domain. Single null mutation of any SacI domain-containing 5-phosphatase shows little phenotype; however, inp51 inp52 and inp52 inp53 double mutants show overlapping functions/phenotype, including actin cytoskeletal disruption, endocytic defects, abnormal cell wall integrity, and vacuolar fragmentation (23.Srinivasan S. Seaman M. Nemoto Y. Daniell L. Suchy S.F. Emr S. De Camilli P. Nussbaum R. Eur. J. Cell Biol. 1997; 74: 350-360PubMed Google Scholar, 24.Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). Deletion of all three SacI domain-containing 5-phosphatases is lethal; interestingly, growth and other defects in the triple mutant can be rescued by the overexpression of mammalian 5-phosphatase II (25.O'Malley C.J. McColl B.K. Kong A.M. Ellis S.L. Wijayaratnam A.P. Sambrook J. Mitchell C.A. Biochem. J. 2001; 355: 805-817Crossref PubMed Scopus (12) Google Scholar). The yeast 5-phosphatase Inp54p contains a catalytic 5-phosphatase domain (26.Raucher D. Stauffer T. Chen W. Shen K. Guo S. York J.D. Sheetz M.P. Meyer T. Cell. 2000; 100: 221-228Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar) but no SacI domain. Inp54p localizes to the ER membrane, and deletion of INP54 results in increased secretion of a mammalian reporter protein bovine pancreatic trypsin inhibitor by ∼2-fold relative to wild-type strains (9.Wiradjaja F. Ooms L.M. Whisstock J.C. McColl B. Helfenbaum L. Sambrook J.F. Gething M.J. Mitchell C.A. J. Biol. Chem. 2001; 276: 7643-7653Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).Several lines of evidence suggest that PtdIns(4,5)P2 may play a significant role in vacuolar function. It has been proposed that PtdIns(4,5)P2 is important for vacuolar fusion, and PtdIns(4,5)P2 is itself synthesized during vacuolar fusion and regulates vacuole ATP-dependent priming and docking (27.Mayer A. Scheglmann D. Dove S. Glatz A. Wickner W. Haas A. Mol. Biol. Cell. 2000; 11: 807-817Crossref PubMed Scopus (100) Google Scholar, 28.Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar). Although PtdIns(4,5)P2 has not yet been identified on vacuolar membranes in intact yeast cells, in vitro studies revealed the recruitment of a PtdIns(4,5)P2 biosensor to the vertices (i.e. the periphery of tightly apposed membranes between docked vacuoles) of vacuoles in docking reactions (28.Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar). In mammalian cells several PtdIns(4,5)P2 lipid phosphatases, including the 5-phosphatase oculocerebrorenal protein of Lowe (OCRL) and the recently identified PtdIns(4,5)P2 4-phosphatases, localize to lysosomal membranes, the mammalian homologue of the vacuole (29.Zhang X. Hartz P.A. Philip E. Racusen L.C. Majerus P.W. J. Biol. Chem. 1998; 273: 1574-1582Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 30.Ungewickell A. Hugge C. Kisseleva M. Chang S.C. Zou J. Feng Y. Galyov E.E. Wilson M. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18854-18859Crossref PubMed Scopus (85) Google Scholar). OCRL is mutated in patients with Lowe syndrome, which includes renal Fanconi syndrome, growth failure, mental retardation, cataracts, and glaucoma (29.Zhang X. Hartz P.A. Philip E. Racusen L.C. Majerus P.W. J. Biol. Chem. 1998; 273: 1574-1582Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Lysosomal hydrolase activity is elevated in plasma from Lowe syndrome patients, relative to age-matched controls (31.Ungewickell A.J. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13342-13344Crossref PubMed Scopus (60) Google Scholar). These studies suggest PtdIns(4,5)P2 levels may be tightly regulated on lysosomal/vacuolar membranes.Previous studies have shown a functional overlap between some phosphoinositide phosphatases in yeast, although not all possible phosphatase interactions have been explored (32.Parrish W.R. Stefan C.J. Emr S.D. Mol. Biol. Cell. 2004; 15: 3567-3579Crossref PubMed Scopus (63) Google Scholar, 33.Stefan C.J. Audhya A. Emr S.D. Mol. Biol. Cell. 2002; 13: 542-557Crossref PubMed Scopus (183) Google Scholar). In this study we have examined the phenotype of mutants lacking both SAC1 and INP54. Inp54p hydrolyzes PtdIns(4,5)P2 to PtdIns(4)P, whereas Sac1p hydrolyzes PtdIns(4)P to PtdIns. We investigated whether these enzymes coordinately regulate PtdIns(4,5)P2 metabolism. We demonstrate here that although the total cellular PtdIns(4,5)P2 levels remain unchanged in sac1 inp54 double mutants, the spatial distribution of PtdIns(4,5)P2 is profoundly altered, accumulating on vacuole membranes. In these double mutants, vacuolar membrane PtdIns(4,5)P2 is derived from the plasma membrane, and its accumulation on a subset of vacuole membranes is associated with defects in vacuolar fusion. These studies reveal tight regulation of PtdIns(4,5)P2 levels at the plasma membrane is required to regulate vacuolar fusion.EXPERIMENTAL PROCEDURESMaterials−All restriction and DNA-modifying enzymes were obtained from Fermentas (Burlington, Canada), New England Biolabs (Beverly, MA), or Promega (Madison, WI). Oligonucleotides were obtained from GeneWorks (Adelaide, Australia). All other reagents were from Sigma or Invitrogen, unless otherwise stated. The constructs pEGFP-N1/PH-PLCδ1 and pEGFP-C1/PH-OSBP were kind gifts from Prof. Tamas Balla, NICHD, National Institutes of Health, Bethesda. GFP-PH-Num1p construct was a gift from Prof. Mark Lemmon, University of Pennsylvania, Philadelphia. Vph1p antibody was from Prof. Tom Stevens, University of Oregon, Eugene. Antibody to Clc1p was a kind gift from Prof. Gregory Payne, UCLA. MFY72 (sac1ts) and AAY202 (mss4ts) strains and constructs encoding MSS4-GFP and GFP-STT4 were donations from Prof. Scott Emr, University of California, San Diego. Yeast strains used in this study are listed in Table 1, and plasmids are listed in Table 2.TABLE 1Yeast strains used in this studyStrainGenotypeSourceSEY6210MATα ura3-52 leu2-3, 112 his3-Δ200 trp-Δ901 lys2-801 suc2-Δ963Δinp54SEY6210, inp54::LEU2This studyΔsac1 (ATY202)SEY6210, sac1::TRP115Δsac1 Δinp54SEY6210, sac1::TRP1, inp54::LEU2This studysac1ts (MFY72)SEY6210, sac1::TRP1, carrying pRS416sac1ts-2341sac1ts Δinp54MFY72, inp54::LEU2This studymss4ts (AAY202)SEY6210, mss4::HIS3MX, carrying YCplac111-mss4ts-10233mss4ts Δsac1 Δinp54AAY202, sac1::TRP1, inp54::URA3This studyΔsac1 Δinp54BY4741 (MATα his3-Δ1 leu2-Δ0 met15-Δ0 ura3-Δ0), sac1::KanMX, inp54::LEU2This studyΔfig4 Δinp54BY4741, fig4::KanMX, inp54::LEU2This studyΔvps55BY4741, vps55::KanMXResGenΔvps27BY4741, vps27::KanMXResGenΔapm3BY4741, apm3::KanMXResGen Open table in a new tab TABLE 2List of plasmids used in this studyPlasmidDescriptionSourceGFP-PH-NUM12μ URA3 vector expressing GFP at the N terminus of PH-NUM144pPS13032μ URA3 vector expressing GFP under a GAL promoter64pPGK1303pPS1303 with the GAL promoter replaced by a PGK promoterThis studypPGK1303/2xPH-PLCδ12xPH-PLCδ1-GFP for localization of PtdIns(4,5)P2This studypPGK1303/PH-OSBPPH-OSBP-GFP for localization of PtdIns(4)PThis studypPGK1303/SEC14SEC14-GFPThis studypPGK-Lys2/2xPH-PLCδ1LYS2 plasmid for localization of PtdIns(4,5)P2 in sac1ts Δinp54 and mss4ts Δsac1 Δinp54 mutantsThis studypRS416-GFP/PIK1CEN URA3 plasmid expressing Pik1p-GFP under native promoterThis studypRS426/GFP-STT42μ URA3 plasmid expressing GFP-Stt4p under native promoterS. EmrpRS426/MSS4-GFP2μ URA3 plasmid expressing Mss4p-GFP under native promoterS. EmrpBINP54URAFor generation of mss4ts Δsac1 Δinp54 strainsThis studypPGK423/HA-INP542μ HIS3 plasmid expressing HA-Inp54p under the PGK promoterThis study Open table in a new tab Disruption of SAC1 and/or INP54−Deletion of SAC1 or INP54 from the SEY6210 strain was as described previously (9.Wiradjaja F. Ooms L.M. Whisstock J.C. McColl B. Helfenbaum L. Sambrook J.F. Gething M.J. Mitchell C.A. J. Biol. Chem. 2001; 276: 7643-7653Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15.Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Double null sac1 inp54 and fig4 inp54 mutants and sac1ts Δinp54 mutants were created by replacing INP54 in the Δsac1, Δfig4 (ResGen/Invitrogen) or MFY72 strains, respectively, with a LEU2 cassette as described previously (9.Wiradjaja F. Ooms L.M. Whisstock J.C. McColl B. Helfenbaum L. Sambrook J.F. Gething M.J. Mitchell C.A. J. Biol. Chem. 2001; 276: 7643-7653Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). This resulted in the deletion of a segment of chr XV from coordinates 206,284–204,565, which spans the whole open reading frame of INP54 from nucleotides 1 to 1155, 400 bp upstream of the start codon and 166 bp downstream of the stop codon. The triple mutant mss4ts Δsac1 Δinp54 was generated by replacing SAC1 with a TRP1 cassette and INP54 with a URA3 cassette in the mss4ts (AAY202) strain. The TRP1 sequence was amplified from pRS424 with the primers 5′-atgacaggtccaatagtgtacgttcaaaatgcggacggtatcttcttcaagcttgctatgtctgttattaatttcag-3′ and 5′-ttaatctctttttaaaggatccggcttggaaaatttaggactgtgcggtatttcacaccg-3′. The PCR product was transformed into mss4ts strains resulting in the deletion of SAC1 from nucleotides 58 to 1830 or chr XI coordinates 34,601–36,373, creating a mss4ts Δsac1 strain. INP54, including 1602 bp upstream of the start codon and 714 bp downstream of the stop codon, was amplified from SEY6210 genomic DNA using the primers 5′-ggctcgagttaaaacgtaagggatatgct-3′ and 5′-gcggccgctgtcgatgtactttatgt-3′ and ligated into an XhoI-NotI-digested pBIIKS(+) to generate pBINP54. URA3, including its promoter, was amplified from pRS426 using the primers 5′-gactagtcgcgcgtttcggtgatgac-3′ and 5′-catgcatttacttataatacag-3′ and cloned into PstI-SpeI-digested pBINP54 to generate pBINP54URA. The URA3 cassette flanked by the sequence upstream and downstream of INP54 was recovered from pBINP54URA by XhoI-NotI digestion, and subsequently transformed into mss4ts Δsac1 strain resulting in the deletion of INP54, including 400 bp upstream and 166 bp downstream of the open reading frame, creating a mss4ts Δsac1 Δinp54 strain. Disruption of each gene was confirmed by PCR with the use of two unique sets of primers.Yeast Immunofluorescence−For the detection of Vph1p, Pep12p, and Clc1p, yeast cells were fixed, spheroplasted, and stained as described previously (9.Wiradjaja F. Ooms L.M. Whisstock J.C. McColl B. Helfenbaum L. Sambrook J.F. Gething M.J. Mitchell C.A. J. Biol. Chem. 2001; 276: 7643-7653Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Anti-Vph1p and anti-Pep12p (Molecular Probes) were detected with anti-mouse Alexa-594 (Molecular Probes) and anti-Clc1p with anti-rabbit Alexa-594 (Molecular Probes). All other observations of GFP-tagged proteins were performed in live yeast cells. Fixed or live cells were placed on poly-l-lysine (2 mg/ml)-coated glass slides, and coverslips were mounted with SlowFade (Molecular Probes).Confocal Microscopy−Yeast cells were visualized and analyzed using either an Olympus Fluoview confocal microscope or a Leica TCS NT confocal microscope, with green fluorescence collected in channel 1 (488 nm excitation, 530 ± 30 nm emission) and red fluorescence in channel 2 (568 nm excitation, LPS90 nm). Images presented in the figures show either cells in a single field or from several different fields.Vacuole Labeling−Yeast cells were grown to mid-log phase and metabolically labeled with 20 μm FM4-64 (Sigma) for 15 min at 28 °C in YPD (34.Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1127) Google Scholar), chased in fresh YPD without FM4-64 for 30–120 min as indicated, and viewed by confocal microscopy. For analysis of endocytosis (Fig. 5), cells were labeled with 2 μm FM4-64 and viewed by confocal microscopy at the indicated time points. For CMAC-Arg labeling, sac1ts Δinp54 cells were incubated with 10 μm CMAC-Arg dye (Molecular Probes) for 4 h at 28 °C, shifted to 38 °C, further incubated for 2 h, and analyzed by confocal microscopy. To assay vacuolar fragmentation or fusion, yeast cells were grown in standard YPD medium, labeled with FM4-64 for 1 h, and then shifted to YPD + 0.4 m NaCl or H2O, respectively, for 30 min. All incubation steps were performed at 28 °C, except for sac1ts Δinp54 cells that were incubated at 38 °C. Approximately 400 cells from three separate experiments were scored for vacuolar fragmentation, expressed as a percentage of the total cell population, and the mean ± S.E. was determined.Analysis of Vacuole Function−YPD agar, pH 8.0, was prepared as described (35.Hongay C. Jia N. Bard M. Winston F. EMBO J. 2002; 21: 4114-4124Crossref PubMed Scopus (52) Google Scholar). Exponentially growing yeast cells were spotted onto agar plates in 10-fold serial dilutions, starting from a cell density of 107 cells/ml. To visualize acidified compartments, yeast cells were incubated with 0.2 mm quinacrine (Sigma) in YPD, pH 8.0, for 5 min at room temperature, as described previously (36.Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar).Inhibition of Endocytosis−To block endocytosis, sac1ts Δinp54 cells expressing 2xPH-PLC-GFP were incubated with 33 μg/ml latrunculin A (Molecular Probes) at 28 °C for 1 h, shifted to 38 °C for 1–2 h, and labeled with 20 μm FM4-64 or 10 μm CMAC-Arg (Molecular Probes).Subcellular Localization of PtdIns(4)P and PtdIns(4,5)P2− PH-OSBP was amplified from pEGFP-C1/PH-OSBP using the primers 5′-ggatccaaacaatgggctcggctcgagagggc-3′ and 5′-ggatccctgccagcatcttcacagc-3′, and the resulting PCR product was cloned into the BglII site of pPGK1303. 2xPH-PLCδ1 was amplified from pEGFP-N1/PH-PLCδ1 using two different sets of primers. The first set incorporated a BamHI site at the 5′ end (5′-ggatccaaacaatggactcgggccgggac-3′) and an EcoRI site at the 3′ end (5′-gaattccttcaggaagttctg-cag-3′), and the second set incorporated an EcoRI site at the 5′ end (5′-gaattcatggactcgggccgggac-3′) and an XhoI site at the 3′ end (5′-ctcgagacttcaggaagttctgcag-3′). The two resulting PCR products were ligated together into BglII-XhoI-digested pPGK1303, and the two PH-PLCδ1 fragments simultaneously ligated via their EcoRI ends, generating two PH-PLC domains in tandem (Table 2). For expression in sac1ts Δinp54 and mss4ts Δsac1 Δinp54 strains, 2xPH-PLC-GFPδ1 was cloned in pPGK-Lys2 vector (Table 2). These constructs were subsequently transformed into yeast cells, and expression of GFP-tagged PH-OSBP, PH-PLCδ1, or PH-Num1p was analyzed live from mid-log phase cultures by confocal microscopy. Δsac1 Δinp54 and sac1ts Δinp54 cells showing 2xPH-PLC-GFP on the vacuole were scored as a percentage of the total cell population, and mean ± S.E. was determined. The proportion of vacuoles expressing 2xPH-PLC-GFP on their membranes relative to the total vacuole number per cell was determined the same way. Data were collected from four independent experiments with at least 400 cells counted.Expression of Sec14p-GFP and Pik1p-GFP−The full 912-bp SEC14 coding sequence without the stop codon (chr XIII coordinates 424,988–426,055) was amplified by PCR from the genomic DNA of SEY6210 strain using the primers 5′-agatctatggttacacaacaagaaaaggaattttta-3′ and 5′-agatctctttcatcgaaaaggcttccgg-3′, incorporating a BglII site at each end. The resulting product was cloned into the BglII site of pPGK1303 (Table 2). The full 3198-bp PIK1 sequence without the stop codon and 1000 bp of upstream sequence (chr XIV coordinates 140,877–144,074) was amplified by PCR using the primers 5′-ggatcctgttccatatctcggtgttgtttg-3′ and 5′-ggatcccgctatatataccctgtgtaataag-3′, incorporating a BamHI site at each end. The product was cloned in the BamHI site of pRS416-GFP (Table 2).Analysis of CPY and General Secretion−Metabolic labeling of CPY was performed by labeling cells with 25 μCi of Easy Tag Trans35S (Amersham Biosciences) per A600 unit at 25 °C for 5 min, followed by chasing in the presence of excess methionine and cysteine for the indicated time periods as described previously (37.Munn A.L. Heese-Peck A. Stevenson B.J. Pichler H. Riezman H. Mol. Biol. Cell. 1999; 10: 3943-3957Crossref PubMed Scopus (139) Google Scholar). CPY was immunoprecipitated using a polyclonal CPY antibody and separated on 8% SDS-PAGE, followed by fluororadiography. CPY secretion was detected using a colony immunoblot assay as described by Roberts et al. (36.Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). The general secretion assay was performed as described previously (38.Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar). Following a 5-min Easy Tag Trans35S labeling and a 30-min chase, NaF and NaN3 were added to the cells to a final concentration of 20 mm each. The cell suspension was centrifuged to collect the medium fraction. Cold trichloroacetic acid was added to a final concentration of 10% to the medium fraction and incubated on ice for 30 min. Following centrifugation, the protein pellet was washed twice with acetone and sonicated in the presence of 2× Laemmli buffer and boiled, and 1 A600 unit was analyzed on 10% SDS-PAGE. General protein secretion was detected by fluororadiography.Analysis of Steady-state Levels of ALP−Extraction of ALP from yeast cells was performed as described previously (39.Belgareh-Touze N. Avaro S. Rouille Y. Hoflack B. Haguenauer-Tsapis R. Mol. Biol. Cell. 2002; 13: 1694-1708Crossref PubMed Scopus (29) Google Scholar). ALP was detected using a monoclonal ALP antibody (Molecular Probes).Analysis of Total Cellular Phosphoinositides by HPLC−[14C]Inositol labeling of yeast, extraction and deacylation of lipids, and HPLC techniques were performed as described previously (15.Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Data were collected from three independent experiments, and the mean ± S.E. was determined.RESULTSTotal Cellular Levels of PtdIns(4,5)P2 Are Unaltered in Δsac1 Δinp54 Mutants−Inp54p and Sac1p may act sequentially to regulate PtdIns(4,5)P2 and PtdIns(4)P levels, re