STT4 Is an Essential Phosphatidylinositol 4-Kinase That Is a Target of Wortmannin in Saccharomyces cerevisiae
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.27671
ISSN1083-351X
AutoresN. Shane Cutler, Joseph Heitman, María E. Cárdenas,
Tópico(s)Transgenic Plants and Applications
ResumoWortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeastSaccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate. Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeastSaccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate. Small cell-permeable compounds have proven valuable tools to study signal transduction. For example, studies on the mechanism of action of the immunosuppressive antifungal natural products cyclosporin A and FK506 led to the identification of the protein phosphatase calcineurin as a critical calcium sensor during T-cell activation and physiological responses in yeast cells. Related studies of another natural product, the immunosuppressant rapamycin, revealed a role for the TOR kinase homologs in regulating cell proliferation in both yeast and mammalian cells (reviewed in Refs. 1Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1963) Google Scholar and 2Cardenas M.E. Lorenz M. Hemenway C. Heitman J. Perspect. Drug Discovery Design. 1994; 2: 103-126Crossref Scopus (31) Google Scholar). Wortmannin is a hydrophobic steroid-related natural product of the fungus Talaromyces wortmannii (reviewed in Ref. 3Ui M. Okada T. Hazeki K. Hazeki O. Trends Biochem. Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (519) Google Scholar). Wortmannin is an immunosuppressive and anti-inflammatory agent that inhibits signal transduction events in a variety of cell types. For example, wortmannin inhibits neutrophil and platelet activation by a variety of ligands, such as leukotriene B4, platelet-activating factor, N-formyl-Met-Leu-Phe, and thromboxane, which act via G-protein-coupled receptors (4Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar, 5Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar). In addition, wortmannin also blocks insulin stimulation of glucose uptake in adipocytes (6Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar). Thus, wortmannin can block signal transduction events emanating from either G-protein-coupled or tyrosine kinase receptors, which signal through distinct pathways. Wortmannin does not affect immediate signal transduction events in these pathways, such as tyrosine phosphorylation, production of inositol trisphosphate (IP3), 1The abbreviations used are: IP3, inositol trisphosphate; PI, phosphatidylinositol; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PI4P, phosphatidylinositol 4-phosphate; PI3P, phosphatidylinositol 3-phosphate; PCR, polymerase chain reaction; PLC, phospholipase C; HA, hemagglutinin; GAL, galactose. 1The abbreviations used are: IP3, inositol trisphosphate; PI, phosphatidylinositol; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PI4P, phosphatidylinositol 4-phosphate; PI3P, phosphatidylinositol 3-phosphate; PCR, polymerase chain reaction; PLC, phospholipase C; HA, hemagglutinin; GAL, galactose. or Ca2+ mobilization. That stimulation of protein kinase C by phorbol ester overcomes the inhibitory effects of wortmannin (7Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar, 8Standaert M.L. Avignon A. Yamada K. Bandyopadhyay G. Farese R.V. Biochem. J. 1996; 313: 1039-1046Crossref PubMed Scopus (60) Google Scholar) suggests that wortmannin acts upstream of protein kinase C, possibly at a point at which G-protein-coupled and tyrosine kinase signaling pathways converge. Several observations suggested that a relevant in vivotarget of wortmannin might be an enzyme, namely the lipid kinase that phosphorylates the 3-position of phosphatidylinositol: PI 3-kinase. First, wortmannin blocks antigen-dependent stimulation of PI 3-kinase activity in rat basophils (4Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar). Second, wortmannin markedly inhibits phosphatidylinositol 3,4,5-trisphosphate production in neutrophils stimulated with N-formyl-Met-Leu-Phe, consistent with a block in phosphorylation of phosphatidylinositol 4,5-bisphosphate (PI4,5P2) by PI 3-kinase (5Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar). Third, the ability of insulin to stimulate PI 3-kinase activity in adipocytes is inhibited by wortmannin (6Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar). In mammalian cells, PI 3-kinase is a heterodimer composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that interacts with other signal transduction elements via SH2 domains (9Kapeller R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar). The activity of purified PI 3-kinase is potently inhibited in vivo and in vitro by wortmannin (4Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar, 5Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 10Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (520) Google Scholar). Finally, using anti-wortmannin antibodies and protease digestion, it has been shown that wortmannin forms a covalent complex with an active site residue of the 110-kDa PI 3-kinase catalytic subunit, lysine 802 (4Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar, 11Wymann M.P. Bulgarelli-Leva G. Zvelebil M.J. Pirola L. Vanhaesebroeck B. Waterfield M.D. Panayotou G. Mol. Cell. Biol. 1996; 16: 1722-1733Crossref PubMed Scopus (633) Google Scholar). Although wortmannin potently inhibits the PI 3-lipid kinase with an IC50 of 5 nm, other potential targets exist. For example, wortmannin inhibits a protein kinase, myosin light chain kinase, with an IC50 of ∼20 nm (12Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar). In addition, wortmannin also inhibits DNA-dependent protein kinase, a member of the PI 3/4-kinase superfamily, with an IC50 of 200 nm (13Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Anderson C.W. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (670) Google Scholar). Demethoxyviridin, a structural analog of wortmannin, inhibits a membrane associated PI 4-kinase from the fission yeast Schizosaccharomyces pombe(IC50 = 100 nm) (14Woscholski R. Kodaki T. McKinnon M. Waterfield M.D. Parker P.J. FEBS Lett. 1994; 342: 109-114Crossref PubMed Scopus (97) Google Scholar), whose identity has not been established. Recently a wortmannin-sensitive, membrane-associated PI 4-kinase, shown previously to maintain hormone-regulated PI4P pools in mammalian cells (15Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (309) Google Scholar, 17Downing G.J. Kim S. Nakanishi S. Catt K.J. Balla T. Biochemistry. 1996; 35: 3587-3594Crossref PubMed Scopus (104) Google Scholar), was cloned from a human cDNA library (18Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Thus, wortmannin inhibits several protein and lipid kinases. Previous studies of antifungal natural products reveal that both the mechanisms of action as well as drug targets are often highly conserved from unicellular to multicellular eukaryotes. We report here our studies on the mechanism of action and targets of wortmannin in the budding yeast Saccharomyces cerevisiae. Our genetic and biochemical evidence indicates that a target of wortmannin in yeast is the PI 4-kinase STT4 (19Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar). Conditions that rescue cells lacking STT4 also overcome wortmannin toxicity, overexpression of STT4 renders cells wortmannin-resistant, and the PI 4-kinase activity of STT4 is inhibited by wortmannin in vitro. The second yeast PI 4-kinase, PIK1 (20Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Abstract Full Text PDF PubMed Google Scholar, 21Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 22Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar), does not appear to be a target of wortmannin in vivo, and the PI 4-kinase activity of PIK1 is not sensitive to wortmannin in vitro. STT4 is tightly associated with a membrane-pellet fraction. In addition, we report that STT4 is essential in some yeast strains. Because wortmannin inhibition causes a nonspecific cell cycle arrest, STT4 function may be required throughout the cell cycle. Mammalian homologs of both yeast STT4 and PIK1 have been recently cloned (15Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). Curiously, the mammalian PIK1 homolog, and not the mammalian STT4 homolog, is wortmannin-sensitivein vitro. We also find that overexpression of the yeast PI4P 5-kinase homolog MSS4 or deletion of the yeast gene encoding phospholipase C (PLC1) confer wortmannin resistance, supporting a model in which the inhibitory effects of wortmannin may be due to depletion of an essential PI4,5P2 pool. Our studies underscore the utility of yeast as a model system to identify drug targets, suggest caution in the use of wortmannin as a specific inhibitor of mammalian PI 3-kinase, reveal that diverse members of the lipid/protein kinase superfamily are wortmannin-sensitive, and provide evidence for a phosphatidylinositol metabolic cascade in yeast involving sequential action of STT4, MSS4, and PLC1 in which PI4,5P2 is an essential lipid product. Strain YS3–6D (MATa leu2 ura3 his3 ade8 met3) and an isogenic stt4 derivative Δstt4–16C (MATa Δstt4::HIS3 leu2 ura3 his3 ade8) were kindly provided by Yoshi Ohya (19Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar). Strain FM1–5d (MATαleu2–3, 112 ura3–52 trp1 his4 ade2Δ GAL + rme1 HMLa) and FM1–5d/pCF12 (pCF12 contains a pik1-ts allele) were a kind gift of Mike Hall (22Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar). Strain SEY6210 (MATa leu2–3, 112 ura3–52 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9) and an isogenic vps34mutant strain PHY102 were from Scott Emr (24Stack J.H. Emr S.D. J. Biol. Chem. 1994; 269: 31552-31562Abstract Full Text PDF PubMed Google Scholar). The STT4 gene was disrupted in the JK9–3da/α diploid strain (ura3–52 leu2–3, 112 his4 trp1 rme1 HMLa) by integrative disruption with the Δstt4::G418 allele obtained by PCR amplification of the G418 resistance gene with primers 5′-AAGGCAGATGAGATTTACCAGAGGATTGAAAGCCTCTTCATCTTTCAGCTGAAGCTTCGTACGC and 5′-GTACGGAATGCCATTTGTGAGCCTTTGGAATTCATCATAACCTTTGCATAGGCCACTAGTGGATCTG with the plasmid pFA6-kanMX2 as template (47Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2235) Google Scholar) to yield theSTT4/Δstt4::G418 strain SMY23–3. Similarly, thePLC1 gene was disrupted in the JK9–3da/α diploid strain by integrative disruption with the Δplc1::G418allele obtained by PCR amplification of the G418 resistance gene with primers 5′-AAACGTACAACGGTAAGGTCATTCACGCAGTGTATATGCAGCTGAAGCTTCGTACGC and 5′-CGTATTTATGAATATGTGTATTTGGCCGGAAAAAGATCGCCGCATAGGCCACTAGTGGATCTG to yield the PLC1/Δplc1::G418 strain SMY31–1. Plasmid-containing Δplc1::G418 andPLC1 haploid strains were obtained by transforming SMY31–1 with the plasmid, sporulating and dissecting. Yeast media were prepared as described (25Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Wortmannin (Sigma) was added to the media from concentrated stocks prepared in Me2SO. DNA manipulations were carried out as outlined in Ref. 26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. Yeast were transformed by the lithium acetate/heat shock method (27Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). The following plasmids were used. 1) pYeF1 and pYeF2 (28Cullin C. Minvielle-Sebastia L. Yeast. 1994; 10: 105-112Crossref PubMed Scopus (66) Google Scholar) are 7.1-kilobase pair 2-μm plasmids carrying the URA3 gene and the GAL promoter and were used to epitope-tag STT4 and PIK1. 2) pGALHA-STT4–8 (HA-STT4) was created by ligating the STT4gene into the NotI site of pYeF1 in frame with the HA epitope tag at the 5′ end of the gene, placing the fusion gene under the control of the GAL promoter. The STT4 gene was amplified by PCR from a plasmid copy of STT4using primers 5′-TTAAGCGGCCGCATGAGATTTACCAGAGGAGATTG and 5′-TATAGCGGCCGCGTCAGTACGGAATGCCATTTGAGCC. 3) pNSC8 (PIK1-HA) was constructed by ligating the PIK1 gene into pYeF2 with the 5′ BamHI site and the 3′ NotI site, in frame with the HA epitope tag at the 3′ end of the gene, placing the fusion gene under the control of the GALpromoter. The PIK1 gene was amplified by PCR from a plasmid copy of PIK1 using primers 5′-TTTTTCGGATCCATGCATAAAGCATCCAGTTCAAAG and 5′-ATTTGGTAGCGGCCGCTATATATACCCTGTGTAATAAG. 4) pNSC19 (2μ FAB1) was constructed by digesting theFAB1 gene, along with approximately 2 kilobase pairs of 5′-untranslated region, from the plasmid pAY60 (29Yamamoto A. DeWald D.B. Boronenkov I.V. Anderson R.A. Emr S.D. Koshland D. Mol. Biol. Cell. 1995; 6: 525-539Crossref PubMed Scopus (234) Google Scholar) usingXhoI and NotI and ligated into the polylinker of the plasmid pRS426 (URA3, 2μ) digested with the same enzymes. 5) pMSS4–12 (2μ MSS4) consists of the MSS4gene on YEp352 (2μ, URA3) (30Yoshida S. Ohya Y. Nakano A. Anraku Y. Mol. Gen. Genet. 1994; 242: 631-640Crossref PubMed Scopus (77) Google Scholar). 6) pEP1 (2μ PLC1) consists of a triple HA epitope cloned into the 5′ end of PLC1 cloned into YEp351 (2μ,LEU2) (31Payne W.E. Fitzgerald-Hayes M. Mol. Cell. Biol. 1993; 13: 4351-4364Crossref PubMed Scopus (79) Google Scholar). For PIK1 assays, cells transformed with plasmid pGALPIK1-HA were inoculated in synthetic media lacking uracil supplemented with 2% raffinose, grown to OD600 = 0.5–0.8, and expression of PIK1-HA induced with 2% galactose for 2 h. As a control for protein expression and antibody specificity, parallel cultures were grown as above and supplemented with 2% glucose instead of galactose. For STT4 assays, the isogenic wild-type STT4strain YS3–6D and the Δstt4–16C strain were grown in YPD media. Preparation of cell extracts, immunoprecipitations, lipid kinase reactions, and TLC analysis were performed as described previously (32Cardenas M.E. Heitman J. EMBO J. 1995; 14: 5892-5907Crossref PubMed Scopus (108) Google Scholar) with the following exceptions. Immunoprecipitations were carried out in equal amounts (by protein) of cell lysate in a total volume of 1 ml with 3 μl of anti-HA mouse monoclonal 12 CAS (Babco) antibody. Where indicated, the immunoprecipitates were preincubated with the appropriate concentration of wortmannin for 10 min at 4 °C, and the reactions were started by addition of 10 μm[γ-32P]ATP and 10 μm MgCl2. All reactions were incubated for 20 min at 30 °C with occasional gentle agitation; under these conditions, the reactions were linear for up to 30 min. Strain JK9–3da transformed with pGAL-HASTT4 was grown in S-raffinose minus uracil media to OD600 = 0.5–0.8, after which 2% galactose was added and incubated for 1 h. Cell-free lysates and P100 and S100 fractions were prepared as described (32Cardenas M.E. Heitman J. EMBO J. 1995; 14: 5892-5907Crossref PubMed Scopus (108) Google Scholar). Cell fractions were analyzed by Western blot with the anti-HA monoclonal antibody (see above), followed by ECL detection (Amersham). Wortmannin is a fungal metabolite that potently inhibits mammalian PI 3-kinase (33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1052) Google Scholar). We sought to identify wortmannin targets in yeast. Wortmannin inhibited the growth of yeast on synthetic minimal medium with a minimum inhibitory concentration of ∼10 μg/ml (Fig.1 A). Wortmannin treatment did not cause cell lysis or a specific cell cycle arrest (data not shown). In addition, wortmannin was not toxic to yeast cells grown on rich YPD media, possibly due to nonspecific drug binding by yeast extract components as has been observed with other toxins. Consistent with this interpretation, addition of yeast extract to synthetic media prevented wortmannin toxicity (data not shown). Wortmannin can inhibit the yeast PI 3-kinase VPS34p in vitro (24Stack J.H. Emr S.D. J. Biol. Chem. 1994; 269: 31552-31562Abstract Full Text PDF PubMed Google Scholar) at concentrations higher (IC50 = 3 μm) than those that inhibit mammalian PI 3-kinase (IC50 ∼ 5 nm) (33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1052) Google Scholar). However, yeast cells lacking VPS34 are both viable and remain wortmannin-sensitive, with a minimum inhibitory concentration of ∼1 μg/ml (Fig. 1 A). Thus, VPS34 is not the only target of wortmannin in yeast. The ∼10-fold increased sensitivity of Δvps34 mutant cells to wortmannin compared with the isogenic VPS34 parent strain could result from an increase in the free drug concentration due to the loss of one drug binding target. Alternatively, this effect may be nonspecific becauseΔvps34 mutant cells have perturbed vacuolar function, grow slowly, and are temperature-sensitive, and thus might be inherently more drug-sensitive. The latter explanation is supported by the finding that end1 and vps3 mutations, which also result in vacuolar defects and temperature-sensitive slow growth, are similarly wortmannin-hypersensitive (data not shown). Because yeast cells lacking VPS34 have no detectable PI 3-kinase activity or PI3P, yet retain sensitivity to wortmannin, this drug must inhibit an additional target other than PI 3-kinase in yeast. Recent studies demonstrate that wortmannin or its analog demethoxyviridin also inhibit PI 4-kinases from mammals and fission yeast (14Woscholski R. Kodaki T. McKinnon M. Waterfield M.D. Parker P.J. FEBS Lett. 1994; 342: 109-114Crossref PubMed Scopus (97) Google Scholar, 16Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (309) Google Scholar, 18Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Two PI 4-kinases, PIK1 and STT4, have been identified in S. cerevisiae (19Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar, 20Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Abstract Full Text PDF PubMed Google Scholar, 21Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 22Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar). We tested whether the PIK1 or STT4 PI 4-kinases are targets for wortmannin in yeast. PIK1 is an essential enzyme (21Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 22Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar). A yeast strain expressing a temperature-sensitive PIK1 mutant (pik1-ts) was inhibited by wortmannin at permissive and semi-permissive temperatures with the same minimum inhibitory concentration as the isogenic wild-typePIK1 strain (Fig. 1 A). These findings suggest that PIK1 is not a target of wortmannin. STT4 is also essential for growth on standard yeast growth media, but the inviability of Δstt4 mutant cells can be remediated on rich media, such as YPD, by 1 m sorbitol (19Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar) and more poorly on a synthetic medium with 1 m sorbitol (Fig.1 B, lower left quadrant). To test if wortmannin inhibits STT4 in vivo, we asked whether 1 msorbitol would rescue cells from wortmannin toxicity. Remarkably, in synthetic complete media, 1 m sorbitol rescued cells from the toxic effects of 10 μg/ml wortmannin (Fig. 1 B). Thus, conditions that allow growth of cells lacking STT4 (Δstt4) also allow growth in the presence of wortmannin. In several other yeast strain backgrounds, we and others 2S. Emr, D. Levin, and D. Voelker, personal communications. have found thatΔstt4 mutant cells are inviable and are not rescued by 1m sorbitol (Fig.2 A and data not shown). Thus, in these other strain backgrounds, STT4 is essential under all conditions. Correspondingly, in strain backgrounds where STT4 is essential, 1 m sorbitol failed to rescue cells from wortmannin toxicity (Fig. 1 B). In summary, in synthetic media lacking sorbitol, STT4 is essential and wortmannin is toxic in all strains. In one unusual strain, both the lethality of anΔstt4 mutation and wortmannin toxicity can be overcome by osmotic support with 1 m sorbitol. These findings provide genetic evidence that the PI 4-kinase STT4 is a target of wortmanninin vivo. STT4 is probably not the only wortmannin-sensitive target in yeast, because sorbitol did not rescue cells lacking STT4 from wortmannin toxicity (Fig. 1 B, lower left quadrants). To explore genetic differences between strain backgrounds with respect to the essential function of STT4 a nonisogenic cross was performed between the Δstt4 viable mutant and our wild-type laboratory strain (in the presence of sorbitol). Sporulation and dissection of 24 tetrads revealed 1 tetrad with two wild-typeSTT4 and two slow growth, sorbitol-dependentΔstt4 segregants (parental ditype), 5 tetrads consisting of two wild-type STT4, one slow growth sorbitol-dependent Δstt4 mutant, and one inviable segregant (tetratype), and 18 tetrads consisting of two wild-type STT4 spores and two inviable spores (nonparental ditype). These results are consistent with a minimum of three unlinked extragenic suppressors required for sorbitol rescue of theΔstt4 mutation. As mentioned above, STT4 is essential in our yeast laboratory strain background, and thus an STT4/Δstt4heterozygous diploid sporulates to yield tetrads containing two viable (STT4) and two inviable (Δstt4) segregants (Fig. 2 A). An HA epitope-tagged version of theSTT4 gene was cloned under the transcriptional control of the GAL promoter (pGALHA-STT4). Expression of STT4 complemented theΔstt4 mutation and restored growth even when cells were grown in glucose, which reduces expression from the GAL promoter (Fig.2 B). Rescue of the stt4::G418 mutant cells required the STT4 expression plasmid, as these cells were inviable on 5-fluoroorotic acid medium, which is toxic to cells containing the plasmid-borne URA3 marker. Interestingly, growth in glucose provided a level of STT4 expression that also conferred resistance to 10 μg/ml wortmannin (Fig.3). Overexpression of STT4 from the GAL promoter markedly inhibited growth, and thus we could not assess wortmannin toxicity under these conditions (data not shown). That STT4 overexpression is toxic suggests the activity of this enzyme may normally be regulated in vivo. Expression from the GAL promoter of an epitope-tagged form of the PIK1 PI 4-kinase, PIK1, complemented a pik1-ts mutation but did not confer wortmannin resistance and was not toxic when cells were grown with either glucose or galactose (Fig. 3 and data not shown). These findings implicate the STT4 PI 4-kinase as a specific target of wortmannin in yeast. To further assess the wortmannin sensitivity of STT4 and PIK1 in vitro, these enzymes were immunoprecipitated with an anti-STT4 polyclonal antiserum or with an HA epitope-tagged version of PIK1 and an anti-HA monoclonal antibody. The immunoprecipitates were incubated with PI and [γ-32P]ATP, and PI4P production was detected by borate thin layer chromatography and autoradiography. Immunoprecipitation from cells expressing STT4 yielded detectable PI 4-kinase activity (Fig. 4 B, lane 2), whereas cells lacking STT4 (Δstt4) yielded no PI 4-kinase activity (Fig. 4 B, lane 4), establishing the specificity of the antisera for STT4. The PI 4-kinase activity of STT4 was inhibited ∼80% and ∼95% by 1 and 10 nm wortmannin, respectively (Fig. 4, B–D). In comparison, galactose induction of PIK1-HA led to a marked overexpression of PIK1 as detected by Western blot (Fig. 4 A) and to a corresponding increase in HA-precipitable PI 4-kinase activity (Fig. 4 B, comparelanes 5 and 7); however, the PI 4-kinase activity of PIK1-HA was completely resistant to wortmannin at a concentration of 1, 10, or 20 μm (Fig. 4 A, lane 6, and data not shown). This is in accord with previous observations that the PI 4-kinase activity of PIK1 purified to homogeneity is not sensitive to wortmannin at high concentrations. 3J. Thorner, personal communication. These findings provide biochemical evidence that the PI 4-kinase activity of STT4 is inhibited by wortmannin, and confirm our genetic evidence that STT4 is a target of wortmannin in yeast. To further characterize STT4, cells expressing HA-STT4 were fractionated into soluble (S100) and insoluble (P100) fractions and the amount of STT4 in these fractions was determined by Western blot. STT4 was exclusively detected in the particulate (P100) fraction (Fig.5). Similar fractionation results were obtained with endogenous STT4 detected with an anti-STT4 polyclonal antisera (data not shown). The strength and nature of the STT4 association to the P100 fraction was tested by treating the cell lysate previous to centrifugation with agents known to disrupt membrane-protein or protein-protein interactions. STT4 was solubilized by 1% SDS; however, treatment with 2% Triton X-100 or with agents that interfere with protein-protein interactions, such as 0.5m NaCl and 1.6 m urea, failed to extract STT4 from the particulate fraction (Fig. 5). These results indicate that STT4 is tightly associated with the pellet fraction, possibly via assoc
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