The Saccharomyces cerevisiae LSB6 Gene Encodes Phosphatidylinositol 4-Kinase Activity
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m207996200
ISSN1083-351X
AutoresGil‐Soo Han, Anjon Audhya, Daniel J. Markley, Scott D. Emr, George Carman,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe LSB6 gene product was identified from the Saccharomyces Genome Data Base (locus YJL100W) as a putative member of a novel type II phosphatidylinositol (PI) 4-kinase family. Cell extracts lacking the LSB6 gene had a reduced level of PI 4-kinase activity. In addition, multicopy plasmids containing the LSB6 gene directed the overexpression of PI 4-kinase activity in cell extracts of wild-type cells, in anlsb6Δ mutant, in a pik1 ts stt4 ts double mutant, and in anpik1 ts stt4 ts lsb6Δ triple mutant. The heterologous expression of theS. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6Δ mutant did not exhibit a growth phenotype and failed to exhibit a defect in phosphoinositide synthesis in vivo, the overexpression of the LSB6 gene could partially suppress the lethal phenotype of an stt4Δ mutant defective in the type IIISTT4-encoded PI 4-kinase indicating that Lsb6p functions as a PI 4-kinase in vivo. Lsb6p was localized to the membrane fraction of the cell, and when overexpressed, GFP-tagged Lsb6p was observed on both the plasma membrane and the vacuole membrane. The enzymological properties (pH optimum, dependence on magnesium or manganese as a cofactor, the dependence of activity on Triton X-100, the dependence on the PI surface concentration, and temperature sensitivity) of the LSB6-encoded enzyme were very similar to the membrane-associated 55-kDa PI 4-kinase previously purified fromS. cerevisiae. The LSB6 gene product was identified from the Saccharomyces Genome Data Base (locus YJL100W) as a putative member of a novel type II phosphatidylinositol (PI) 4-kinase family. Cell extracts lacking the LSB6 gene had a reduced level of PI 4-kinase activity. In addition, multicopy plasmids containing the LSB6 gene directed the overexpression of PI 4-kinase activity in cell extracts of wild-type cells, in anlsb6Δ mutant, in a pik1 ts stt4 ts double mutant, and in anpik1 ts stt4 ts lsb6Δ triple mutant. The heterologous expression of theS. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6Δ mutant did not exhibit a growth phenotype and failed to exhibit a defect in phosphoinositide synthesis in vivo, the overexpression of the LSB6 gene could partially suppress the lethal phenotype of an stt4Δ mutant defective in the type IIISTT4-encoded PI 4-kinase indicating that Lsb6p functions as a PI 4-kinase in vivo. Lsb6p was localized to the membrane fraction of the cell, and when overexpressed, GFP-tagged Lsb6p was observed on both the plasma membrane and the vacuole membrane. The enzymological properties (pH optimum, dependence on magnesium or manganese as a cofactor, the dependence of activity on Triton X-100, the dependence on the PI surface concentration, and temperature sensitivity) of the LSB6-encoded enzyme were very similar to the membrane-associated 55-kDa PI 4-kinase previously purified fromS. cerevisiae. PI 1The abbreviations used are: PI, phosphatidylinositol; GFP, green fluorescent protein; Ni-NTA, nickel-nitrilotriacetic acid; HA, hemagglutinin; CDTA, 1,2-cyclohexylenedinitrilotetraacetic acid; CMAC, 7-amino-4-chloromethylcoumarin 4-kinase (ATP:phosphatidylinositol-4-phosphotransferase, EC 2.7.1.67) catalyzes the formation of PI 4-phosphate and ADP from PI and ATP (1Colodzin M. Kennedy E.P. J. Biol. Chem. 1965; 240: 3771-3780Google Scholar). The PI 4-phosphate product of the reaction serves as the precursor to the polyphosphoinositides PI 4,5-bisphosphate, PI 3,4-bisphosphate, and PI 3,4,5-trisphosphate (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Google Scholar, 3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar, 4Gehrmann T. Heilmayer Jr., L.G. Eur. J. Biochem. 1998; 253: 357-370Google Scholar). The synthesis and turnover of these polyphosphoinositides have received a great deal of attention because of their roles in receptor-mediated signal transduction, vesicle trafficking, endocytosis, and cytoskeletal reorganization (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Google Scholar, 3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar, 4Gehrmann T. Heilmayer Jr., L.G. Eur. J. Biochem. 1998; 253: 357-370Google Scholar, 5De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Google Scholar). Two types of PI 4-kinase enzymes (types II and III) have been identified in mammalian cells and in the yeast Saccharomyces cerevisiaebased on their biochemical properties (3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar, 4Gehrmann T. Heilmayer Jr., L.G. Eur. J. Biochem. 1998; 253: 357-370Google Scholar, 6Carman G.M. Buxeda R.J. Nickels Jr., J.T. Gross R.W. Advances in Lipobiology. Jai Press Inc., Greenwich, CT1996: 367-385Google Scholar). Genes (or cDNAs) encoding the type III PI 4-kinase enzymes have been identified from mammalian cells and yeast (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Google Scholar, 3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar, 7Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Google Scholar). They all contain a common catalytic kinase domain, which is also found in the type I PI 3-kinase family of enzymes (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Google Scholar, 3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar, 7Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Google Scholar). Until recently (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar, 9Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Google Scholar), a cDNA encoding a type II PI 4-kinase had not been identified. In fact, the elusive nature of this identification has led to the assumption that type II PI 4-kinase enzymes may be proteolytic fragments or splice variants of the type III PI 4-kinase enzymes (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar, 10Flanagan C.A. Schnieders E.S. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Google Scholar). However, advances in the sensitivity of protein sequence methodology have facilitated the identification and the cloning of human (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar) and rat brain (9Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Google Scholar) cDNAs encoding type II PI 4-kinase enzymes. The predicted protein sequences of the type II PI 4-kinase enzymes lack the characteristic catalytic kinase domain found in the type I PI 3-kinase and type III PI 4-kinase enzymes (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar, 9Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Google Scholar). The human and rat brain type II PI 4-kinase enzymes represent the founding members of a novel family of PI 4-kinase enzymes that are highly conserved throughout evolution (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar, 9Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Google Scholar). PIK1 (10Flanagan C.A. Schnieders E.S. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Google Scholar) and STT4 (11Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Google Scholar) are essential genes inS. cerevisiae that encode for type III PI 4-kinase enzymes. The PIK1-encoded PI 4-kinase is a soluble 125-kDa enzyme (12Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Google Scholar), whereas the STT4-encoded PI 4-kinase is a membrane-associated 214.6-kDa enzyme (11Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Google Scholar). No other genes encoding PI 4-kinase enzymes have been identified from yeast. Moreover, the synthesis of PI 4-phosphate and PI 4,5-bisphosphate in a temperature-sensitive pik1 ts stt4 ts double mutant shifted to the restrictive temperature is reduced by 90–95% (13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google Scholar). Accordingly, it has been suggested that the PIK1-encoded and STT4-encoded enzymes may represent the only PI 4-kinase enzymes in yeast (13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google Scholar). However, biochemical evidence indicates the presence of additional PI 4-kinase enzymes in S. cerevisiae. Two membrane-associated forms (55- and 45-kDa) of PI 4-kinase have been purified and characterized from yeast (6Carman G.M. Buxeda R.J. Nickels Jr., J.T. Gross R.W. Advances in Lipobiology. Jai Press Inc., Greenwich, CT1996: 367-385Google Scholar, 14Belunis C.J. Bae-Lee M. Kelley M.J. Carman G.M. J. Biol. Chem. 1988; 263: 18897-18903Google Scholar, 15Buxeda R.J. Nickels Jr., J.T. Belunis C.J. Carman G.M. J. Biol. Chem. 1991; 266: 13859-13865Google Scholar, 16Nickels Jr., J.T. Buxeda R.J. Carman G.M. J. Biol. Chem. 1992; 267: 16297-16304Google Scholar, 17Buxeda R.J. Nickels Jr., J.T. Carman G.M. J. Biol. Chem. 1993; 268: 6248-6255Google Scholar, 18Nickels Jr., J.T. Buxeda R.J. Carman G.M. J. Biol. Chem. 1994; 269: 11018-11024Google Scholar). These enzymes have been classified as type II-like PI 4-kinases (3Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Google Scholar). The deduced protein sequence of LSB6 (Lasseventeen binding), a gene of unknown function in the S. cerevisiae data base, has been identified as a putative member of the novel type II PI 4-kinase family (8Minogue S. Anderson J.S. Waugh M.G. Dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Google Scholar, 9Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Google Scholar). TheLSB6 gene was originally identified in a two-hybrid screen using Las17p/Bee1p as bait (19Madania A. Dumoulin P. Grava S. Kitamoto H. Scharer-Brodbeck C. Soulard A. Moreau V. Winsor B. Mol. Biol. Cell. 1999; 10: 3521-3538Google Scholar). The Las17p/Bee1p protein plays a role in actin patch assembly and actin polymerization (20Li R. J. Cell Biol. 1997; 136: 649-658Google Scholar, 21Lechler T. Li R. J. Cell Biol. 1997; 138: 95-103Google Scholar). In this work, we showed that the LSB6 gene encodes a membrane-associated PI 4-kinase. The enzyme, which was not essential for cell growth under common laboratory conditions, could partially suppress the lethal phenotype of a mutant defective in theSTT4-encoded PI 4-kinase. The LSB6-encoded enzyme was localized to the plasma membrane and vacuolar membrane, and possessed enzymological properties similar to that of the membrane-associated 55-kDa PI 4-kinase enzyme. All chemicals were reagent grade. Growth medium supplies were from Difco. Restriction enzymes, modifying enzymes, and vent DNA polymerase were from New England Biolabs. PCR and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. The Prism DyeDeoxy DNA sequencing kit was from Applied Biosystems. The Yeastmaker yeast transformation system was from Clontech. The DNA size ladder used for agarose gel electrophoresis was from Invitrogen. The plasmid DNA purification and DNA gel extraction kits and Ni-NTA-agarose were from Qiagen, Inc. Phenylmethylsulfonyl fluoride, bovine serum albumin, benzamidine, aprotinin, leupeptin, pepstatin, polyvinylpyrrolidone, and Triton X-100 were purchased from Sigma. Lipids were obtained from Avanti Polar Lipids. Silica Gel 60 thin layer chromatography plates were from EM Science. Radiochemicals and Tran35S-label were purchased from PerkinElmer Life Sciences. Protein assay reagents, electrophoretic reagents, immunochemical reagents, and molecular mass standards were purchased from Bio-Rad. Mouse monoclonal anti-HA antibodies (12CA5) and goat anti-mouse IgG alkaline phosphatase conjugates were purchased from Roche Molecular Biochemicals and Pierce, respectively. Anti-GFP antiserum was obtained from Charles Zuker (University of California, San Diego). Protein A-Sepharose CL-4B beads, polyvinylidene difluoride membrane, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. Scintillation counting supplies and acrylamide solutions were purchased from National Diagnostics. CellTrackerTM Blue CMAC was purchased from Molecular Probes. The strains used in this work are listed in Table I. Methods for yeast growth were performed as described previously (22Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Yeast cells were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete (SC) medium containing 2% glucose at 30 °C. For selection of cells bearing plasmids, appropriate amino acids were omitted from SC medium. Escherichia coli strain DH5α was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (100 μg/ml) was added to bacterial cultures carrying plasmids. Media were supplemented with either 2 (yeast) or 1.5% (E. coli) agar for growth on plates. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm.Table IStrains used in this workStrainRelevant characteristicsSource or Ref.E. coliDH5αF−φ80dlacZΔM15 Δ(lacZYA-argF)U169deoR, recA1 endA1hsdR17(r k− m k+) phoAsupE44 λ− thi-1 gyrA96relA123Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google ScholarBL21(DE3)pLysF− ompT hsdS B(r B− m B−)gal dcm (DE3) pLysSNovagenS. cerevisiaeW303–1AMAT a ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–160Thomas B. Rothstein R. Cell. 1989; 56: 619-630Google ScholarSEY6210MATα his3-Δ200 leu2–3,112 lys2-Δ801 suc2-Δ9 trp1-Δ901 ura3–5261Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Google ScholarSEY6210.1MAT a his3-Δ200 leu2–3,112 lys2-Δ801 suc2-Δ9 trp1-Δ901 ura3–5261Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Google ScholarSEY6210a/αMAT a /MATα his3-Δ200/his3-Δ200 leu2–3,112/leu2–3,112 lys2-Δ801/lys2-Δ801 suc2-Δ9/suc2-Δ9 trp1-Δ901/trp1-Δ900 ura3–52/ura3–5261Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Google ScholarAAY102.1stt4Δ∷HIS3 derivative of SEY6210.1 carrying pRS415-stt4–4 ts13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google ScholarAAY104pik1Δ∷HIS3 derivative of SEY6210 carrying pRS314-pik1–83 ts13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google ScholarAAY104.1pik1Δ∷HIS3 derivative of SEY6210.1 carrying pRS314-pik1–83 ts13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google ScholarAAY105pik1Δ∷HIS3 stt4Δ∷HIS3 derivative of SEY6210 carrying pRS314-pik1–83 ts and pRS415-stt4–4 ts13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google ScholarAAY313lsb6Δ∷HIS3derivative of SEY6210This studyAAY314lsb6Δ∷HIS3 stt4Δ∷HIS3 derivative of SEY6210.1 carrying pRS415-stt4–4 tsThis studyAAY315pik1Δ∷HIS3 lsb6Δ∷HIS3 derivative of SEY6210 carrying pRS314-pik1–83 tsThis studyAAY317pik1Δ∷HIS3 lsb6Δ∷HIS3 stt4Δ∷HIS3 derivative of SEY6210 carrying pRS314-pik1–83 ts and pRS415-stt4–4 tsThis studyAAY1179LSB6-GFP∷HIS3MX6 derivative of SEY6210This studyAAY101stt4Δ∷HIS3/STT4 derivative of SEY6210a/α13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google ScholarAAY103pik1Δ∷HIS3/PIK1 derivative of SEY6210a/α13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google Scholar Open table in a new tab For heterologous expression of the LSB6 gene product,E. coli strain BL21(DE3)pLys bearing plasmid pGH304 was grown at 30 °C in 1000 ml of LB media containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). When the cell density reached an A 600nm of 0.5, the expression of theLSB6 gene was induced by the addition of 1 mmisopropyl-β-d-thiogalactoside to the growth medium. After incubation for 6 h, the induced cells were harvested by centrifugation at 5,000 × g for 5 min at 4 °C. Plasmid and genomic DNA preparation, restriction enzyme digestions, and DNA ligations were performed by standard methods (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Conditions for the amplification of DNA by PCR were optimized as described previously (24Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego1990: 3-12Google Scholar). Transformation of yeast (25Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Google Scholar, 26Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Google Scholar) andE. coli (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) was performed as described previously. DNA sequencing reactions were performed by the dideoxy method usingTaq polymerase (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The plasmids used in this work are listed in Table II. The LSB6 gene (locus YJL100W) (NCBI accession number 6322361) was cloned by PCR. A 3.3-kb DNA fragment containing 1 kb of the 5′-untranslated region, the entire protein coding sequence (1.8 kb), and 0.5 kb of the 3′-flanking sequence was obtained by PCR (primers: 5′-CAAGAGCTCGATACCTTCATCCCTTATG-3′ and 5′-TGCTAGACACTCACCAAACTGCCTTTCC-3′) using strain W303-1A genomic DNA as a template. This LSB6 DNA fragment was digested withSacI/DraI and inserted intoSacI/SmaI sites of plasmid YEp351 to generate plasmid pGH301. LSB6 HA contains sequences for an HA epitope tag inserted after the start codon. The LSB6 PCR DNA fragment was used as a template to produce a 5′-fragment ofLSB6 HA (primers: 5′-CAAGAGCTCGATACCTTCATCCCTTATG-3′ and 5′-AGCGTAGTCTGGGACGTCGTATGGGTACATGCTTTGGTTGGCTTCTTGAAAGTGTCT-3′) and a 3′-fragment of LSB6 HA (primers: 5′-TACCCATACGACGTCCCAGACTACGCTAGTAACGAAGCTTACCAGCATGATCATACC-3′ and 5′-TGCTAGACACTCACCAAACTGCCTTTCC-3′). The 5′- and 3′-fragments ofLSB6 HA were digested withSacI/AatII and AatII/DraI and inserted into the SacI/SmaI sites of YEp351 to generate plasmid pDH1. LSB6 andLSB6 HA DNA fragments were released from plasmids pGH301 and pDH1 by SacI/PstI digestion and inserted into the same restriction sites of plasmid YEp352 to generate plasmids pGH302 and pGH303, respectively. The 1.8-kb LSB6open reading frame was amplified by PCR using plasmid pGH301 as the template (primers: 5′-CGGGATCCGATGAGTAACGAAGCTTACCAG-3′ and 5′-CGCGGATCCTCAACACCAGGTGAATACGGG-3′). The PCR product was digested with BamHI and inserted into the same restriction site in plasmid pET-15b to generate plasmid pGH304. The correct in-frame fusion was confirmed by restriction enzyme analysis. Plasmid constructions were confirmed by DNA sequencing. To generate a GFP-tagged form of Lsb6p, the GFP open reading frame was integrated at theLSB6 locus just upstream of the stop codon to generate AAY1179 as described in Longtine et al. (27Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Google Scholar). A 1.7-kb fragment of LSB6 fused to GFP was amplified by PCR using genomic DNA from AAY1179 as the template (primers: 5′-GAGAAACACAGATAGAGGTCTAGACAATTG-3′ and 5′-GCTCTAGACTCGACCCATGGAGTCTAGAATTCCACCATATTACCCTGTTATCCCTAGCGGATCTGC-3′). The PCR product was digested with XbaI and inserted into the same restriction site in plasmid pGH301 to generate pJA372. Plasmids pGH301, pDH1, pGH302, pGH303, and pJA372 were transformed into the indicated S. cerevisiae mutants for the overexpression of the LSB6 gene product. Plasmid pGH304 was transformed intoE. coli for the recombinant expression of the His-taggedLSB6 gene product.Table IIPlasmids used in this workPlasmidRelevant characteristicsSource or Ref.YEp351MulticopyE. coli/yeast shuttle vector containingLEU262Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Google ScholarYEp352Multicopy E. coli/yeast shuttle vector containing URA362Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Google ScholarpGH301LSB6 gene ligated into theSacI/SmaI sites of YEp351This studypGH302LSB6 gene ligated into theSacI/SmaI sites of YEp352This studypDH1HA-tagged LSB6 gene ligated into theSacI/SmaI sites of YEp351This studypGH303HA-tagged LSB6 gene ligated into theSacI/SmaI sites of YEp352This studypET-15bE. coli expression vector under T7lac promoter for N-terminal His tag fusionNovagenpGH304LSB6 ORF ligated into the BamHI site of pET-15bThis studypJA372LSB6-GFP ligated into the XbaI site of pGH301This study Open table in a new tab A disruption cassette containing 0.9 kb of HIS3 flanked by 50 bp of the 5′-untranslated sequence and 50 bp of the 3′-flanking sequence of theLSB6 gene was generated by PCR (primers: 5′-TATAACCGGGCATAAAGTGAACTAGACACTTTCAAGAAGCCAACCAAAGCCTCTTGGCCTCCTCTAG-3′ and 5′-GAGTTATGATTTCTTTATATTGAGTATGTATTGAATTATTTTCCAAAAAATCGTTCAGAATGACACG-3′). The PCR product was transformed into strain SEY6210 to delete the chromosomal copy of the LSB6 gene by the one-step gene replacement technique (28Rothstein R. Methods Enzymol. 1991; 194: 281-301Google Scholar). Transformants were selected for their ability to grow on SC medium without histidine. The deletion of theLSB6 gene was confirmed by PCR amplification of a 1.3-kb genomic DNA fragment using primers for LSB6(5′-CTGCTCGATACCTTCATCCCTTATGTGTTC-3′) and HIS3(5′-CCCTTTAAAGAGATCGCAATCTGA-3′). One of the lsb6Δ mutants that we isolated was designated strain AAY313. The pik1 ts stt4 ts double mutant (strain AAY105) was generated by mating pik1 ts (strain AAY104) andstt4 ts (strain AAY102.1) followed by tetrad dissection. The pik1 ts stt4 ts lsb6Δ triple mutant (strain AAY317) was generated by mating stt4 ts lsb6Δ (strain AAY314) with pik1 ts lsb6Δ (strain AAY315) and subsequent tetrad dissection. The stt4 ts lsb6Δ (strain AAY314) and the pik1 ts lsb6Δ (strain AAY315) double mutants were generated by matingstt4 ts (strain AAY102.1) with lsb6Δ (strain AAY313) and mating pik1 ts (strain AAY104.1) with lsb6Δ (strain AAY313). Thepik1 ts stt4 ts lsb6Δ triple mutant was confirmed by PCR. All steps were performed at 4 °C. Cells grown to exponential phase were harvested by centrifugation and disrupted with glass beads with a Mini-Bead Beater (Biospec Products) in 50 mm Tris-maleate buffer (pH 7.0) containing 1 mmNa2EDTA, 0.3 m sucrose, 10 mm2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, and 5 μg/ml each of aprotinin, leupeptin, and pepstatin (29Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Google Scholar). The homogenate was centrifuged for 10 min at 1,500 × g to remove glass beads and unbroken cells, and the resulting supernatant was used as the cell extract. The cell extract was centrifuged at 100,000 × g for 1 h to obtain the soluble (supernatant) and total membrane (pellet) fractions (30Lin Y.-P. Carman G.M. J. Biol. Chem. 1989; 264: 8641-8645Google Scholar). The membranes were suspended in Tris-maleate buffer (pH 7.0) containing 10 mm 2-mercaptoethanol and 20% glycerol. Protein concentration was determined by the method of Bradford (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as the standard. Membranes were suspended in Tris-maleate buffer (pH 7.0) containing 10 mm 2-mercaptoethanol, 20% glycerol, and 1 mNaCl at a final protein concentration of 10 mg/ml. The suspension was then centrifuged at 100,000 × g for 1 h to obtain the salt-extractable membrane protein fraction (supernatant). Alternatively, membranes were suspended in the same buffer except that 1% Triton X-100 was substituted for NaCl. The suspension was incubated for 1 h on a shaker. After the incubation, the suspension was centrifuged at 100,000 × g for 1 h to obtain the Triton X-100-extractable membrane protein fraction (supernatant). All steps were performed at 4 °C. E. coli cells containing the His-tagged LSB6-encoded PI 4-kinase were washed once in 20 mm Tris-HCl (pH 8.0) buffer and suspended in 30 ml of 20 mm Tris-HCl (pH 8.0) buffer containing 0.5 mNaCl, 5 mm imidazole, and 1 mmphenylmethylsulfonyl fluoride. Cells were disrupted by a freeze-thaw cycle followed by two passes through a French press at 20,000 pounds/square inch. Unbroken cells and cell debris were removed by centrifugation at 12,000 × g for 30 min at 4 °C. The cell extract (supernatant) was gently mixed for 2 h with 2 ml of 50% slurry of Ni-NTA-agarose. The enzyme/Ni-NTA-agarose mixture was packed in a small disposable column. The column was washed with 10 ml of 20 mm Tris-HCl (pH 8.0) buffer containing 0.5m NaCl, 5 mm imidazole, and 1 mmphenylmethylsulfonyl fluoride and then with 30 ml of 20 mmTris-HCl (pH 8.0) buffer containing 0.5 m NaCl, 45 mm imidazole, 10% glycerol, and 7 mm2-mercaptoethanol. The His-tagged enzyme was then eluted from the column in 1-ml fractions with a total of 4 ml of 20 mmTris-HCl (pH 8.0) buffer containing 0.5 m NaCl, 250 mm imidazole, 10% glycerol, and 7 mm2-mercaptoethanol. SDS-PAGE (32Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) using 10% slab gels and immunoblotting (33Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Google Scholar) using polyvinylidene difluoride membranes were performed as described previously. Mouse monoclonal anti-HA antibodies were used at a final protein concentration of 1 μg/ml. Goat anti-mouse IgG-alkaline phosphatase conjugate was used as a secondary antibody at a dilution of 1:5000. The HA-taggedLSB6-encoded PI 4-kinase was detected on immunoblots using the enhanced chemifluorescence Western blotting detection kit as described by the manufacturer. The HA-tagged protein on immunoblots was acquired by FluorImaging analysis. The relative density of the protein was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability. Cell labeling and immunoprecipitations were performed as described previously (13Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Google Scholar). Briefly, exponential phase cells were concentrated and labeled with Tran35S-Label for 10 min in yeast nitrogen base. Cells were then chased with 5 mm methionine, 2 mmcysteine, and 0.2% yeast extract for the indicated times, and proteins were precipitated with 9% trichloroacetic acid. Lsb6p-GFP was immunoprecipitated from cell extracts with anti-GFP antibodies. Immunoprecipitated proteins were subjected to SDS-PAGE and fluorography. Labeling cells with the vital dye CMAC was performed essentially as described by Stefan et al.(34Stefan C.J. Audhya A. Emr S.D. Mol. Biol. Cell. 2002; 13: 542-557Google Scholar). Briefly, cells were grown to early exponential phase in yeast nitrogen base containing appropriate amino acids and concentrated by centrifugation. Cells were then labeled with 100 nm CMAC in YEPD, followed by a chase in YPED without the vital dye for 45 min. Cells were concentrated by centrifugation and visualized by fluorescence microscopy using an Axiovert S1002TV inverted fluorescent microscope. Images were subsequently processed using a Delta Vision deconvolution system. PI 4-kinase activity was measured for 10 min by following the phosphorylation of 0.2 mm PI with 2.5 mm[γ-32P]ATP (10,000 cpm/nmol) in the presence of 50 mm Tris-maleate buffer (pH 7.0), 3.2 mm Triton X-100, 10 mm MgCl2, and enzyme protein in a total volume of 0.1 ml (16Nickels Jr., J.T. Buxeda R.J. Carman G.M. J. Biol. Chem. 1992; 267: 16297-16304Google Scholar). The reaction was terminated by the addition of 0.5 ml of 0.1 n HCl in methanol. The32P-labeled phospholipid product of the PI 4-kinase reaction was extracted with chloroform (35Carman G.M. Belunis C.J. Nickels Jr., J.T. Methods Enzymol. 1992; 209: 183-189Google Scholar) and analyzed by chromatography on EDTA-treated silica gel plates (36Steiner S. Lester R.L. J. Bacteriol. 1972; 109: 81-88Google Scholar) using solvent system A and by chromatography on CDTA-treated silica gel plates (37Walsh J.P. Caldwell K.K. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9184-9187Google Scholar) using solvent system B with PI 4-phosphate and PI 3-phosphate standards. The PI 3-phosphate standard was produced from a PI 3-kinase reaction as described previously (38Stack J.H. Emr S.D. J. Biol. Chem. 1994; 269: 31552-31562Google Scholar). Solvent system A (36Steiner S. Lester R.L. J. Bacteriol. 1972; 109: 81-88Google Scholar) contained chloroform, methanol, 2.5 m ammonium hydroxide (9:7:2, v/v) (36Steiner S. Lester R.L. J. Bacteriol. 1972; 109: 81-88Google Scholar) a
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