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J. Biol. Chem., Vol. 277, Issue 49, 47709-47718, December 6, 2002

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The Saccharomyces cerevisiae LSB6 Gene Encodes Phosphatidylinositol 4-Kinase Activity^*

Gil-Soo Han, Anjon Audhya^§, Daniel J. Markley, Scott D. Emr^§¶, and George M. Carman

From the  Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901 and the ^§ Department of Cellular and Molecular Medicine, and The Howard Hughes Medical Institute, University of California School of Medicine, San Diego, La Jolla, California 92093

Received for publication, August 6, 2002, and in revised form, October 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 an lsb6 mutant, in a pik1^ts stt4^ts double mutant, and in an pik1^ts stt4^ts lsb6 triple mutant. The heterologous expression of the S. cerevisiae LSB6 gene in Escherichia coli resulted in the expression of a protein that possessed PI 4-kinase activity. Although the lsb6 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 stt4 mutant defective in the type III STT4-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 from S. cerevisiae.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PI¹ 4-kinase (ATP:phosphatidylinositol-4-phosphotransferase, EC 2.7.1.67) catalyzes the formation of PI 4-phosphate and ADP from PI and ATP (1). 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 (2-4). 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 (2-5). Two types of PI 4-kinase enzymes (types II and III) have been identified in mammalian cells and in the yeast Saccharomyces cerevisiae based on their biochemical properties (3, 4, 6). Genes (or cDNAs) encoding the type III PI 4-kinase enzymes have been identified from mammalian cells and yeast (2, 3, 7). They all contain a common catalytic kinase domain, which is also found in the type I PI 3-kinase family of enzymes (2, 3, 7).

Until recently (8, 9), 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 (8, 10). However, advances in the sensitivity of protein sequence methodology have facilitated the identification and the cloning of human (8) and rat brain (9) 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 (8, 9). 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 (8, 9).

PIK1 (10) and STT4 (11) are essential genes in S. cerevisiae that encode for type III PI 4-kinase enzymes. The PIK1-encoded PI 4-kinase is a soluble 125-kDa enzyme (12), whereas the STT4-encoded PI 4-kinase is a membrane-associated 214.6-kDa enzyme (11). 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% (13). Accordingly, it has been suggested that the PIK1-encoded and STT4-encoded enzymes may represent the only PI 4-kinase enzymes in yeast (13). 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 (6, 14-18). These enzymes have been classified as type II-like PI 4-kinases (3).

The deduced protein sequence of LSB6 (Las seventeen 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 (8, 9). The LSB6 gene was originally identified in a two-hybrid screen using Las17p/Bee1p as bait (19). The Las17p/Bee1p protein plays a role in actin patch assembly and actin polymerization (20, 21). 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 the STT4-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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 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. CellTracker^TM Blue CMAC was purchased from Molecular Probes.

Strains and Growth Conditions-- The strains used in this work are listed in Table I. Methods for yeast growth were performed as described previously (22, 23). 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 DH5 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.

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 the LSB6 gene was induced by the addition of 1 mM isopropyl--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.

DNA Manipulations, Amplification of DNA by PCR, and DNA Sequencing-- Plasmid and genomic DNA preparation, restriction enzyme digestions, and DNA ligations were performed by standard methods (23). Conditions for the amplification of DNA by PCR were optimized as described previously (24). Transformation of yeast (25, 26) and E. coli (23) was performed as described previously. DNA sequencing reactions were performed by the dideoxy method using Taq polymerase (23).

Construction of Plasmids Bearing the LSB6 Gene-- 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 with SacI/DraI and inserted into SacI/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 of LSB6^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 of LSB6^HA were digested with SacI/AatII and AatII/DraI and inserted into the SacI/SmaI sites of YEp351 to generate plasmid pDH1. LSB6 and LSB6^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 LSB6 open 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 the LSB6 locus just upstream of the stop codon to generate AAY1179 as described in Longtine et al. (27). 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 into E. coli for the recombinant expression of the His-tagged LSB6 gene product.

Construction of the lsb6 Mutant-- 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 the LSB6 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 (28). Transformants were selected for their ability to grow on SC medium without histidine. The deletion of the LSB6 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 lsb6 mutants that we isolated was designated strain AAY313.

Construction of the pik1^ts stt4^ts Double Mutant and the pik1^ts stt4^ts lsb6 Triple Mutant-- The pik1^ts stt4^ts double mutant (strain AAY105) was generated by mating pik1^ts (strain AAY104) and stt4^ts (strain AAY102.1) followed by tetrad dissection. The pik1^ts stt4^ts lsb6 triple mutant (strain AAY317) was generated by mating stt4^ts lsb6 (strain AAY314) with pik1^ts lsb6 (strain AAY315) and subsequent tetrad dissection. The stt4^ts lsb6 (strain AAY314) and the pik1^ts lsb6 (strain AAY315) double mutants were generated by mating stt4^ts (strain AAY102.1) with lsb6 (strain AAY313) and mating pik1^ts (strain AAY104.1) with lsb6 (strain AAY313). The pik1^ts stt4^ts lsb6 triple mutant was confirmed by PCR.

Preparation of Subcellular Fractions and Protein Determination-- 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 mM Na₂EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 5 µg/ml each of aprotinin, leupeptin, and pepstatin (29). 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 (30). 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 (31) using bovine serum albumin as the standard.

Preparation of NaCl and Triton X-100 Extracts-- Membranes were suspended in Tris-maleate buffer (pH 7.0) containing 10 mM 2-mercaptoethanol, 20% glycerol, and 1 M NaCl 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).

Purification of LSB6-encoded PI 4-Kinase from E. coli-- 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 M NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl 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 0.5 M NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride and then with 30 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 0.5 M NaCl, 45 mM imidazole, 10% glycerol, and 7 mM 2-mercaptoethanol. The His-tagged enzyme was then eluted from the column in 1-ml fractions with a total of 4 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 0.5 M NaCl, 250 mM imidazole, 10% glycerol, and 7 mM 2-mercaptoethanol.

SDS-PAGE and Immunoblotting-- SDS-PAGE (32) using 10% slab gels and immunoblotting (33) 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-tagged LSB6-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.

Metabolic Labeling and Immunoprecipitation-- Cell labeling and immunoprecipitations were performed as described previously (13). 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 mM cysteine, 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.

Fluorescence Microscopy-- Labeling cells with the vital dye CMAC was performed essentially as described by Stefan et al. (34). 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.

Enzyme Assays, Product Identification, and Analysis of Kinetic Data-- PI 4-kinase activity was measured for 10 min by following the phosphorylation of 0.2 mM PI with 2.5 mM [-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 (16). The reaction was terminated by the addition of 0.5 ml of 0.1 N HCl in methanol. The ³²P-labeled phospholipid product of the PI 4-kinase reaction was extracted with chloroform (35) and analyzed by chromatography on EDTA-treated silica gel plates (36) using solvent system A and by chromatography on CDTA-treated silica gel plates (37) 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 (38). Solvent system A (36) contained chloroform, methanol, 2.5 M ammonium hydroxide (9:7:2, v/v) (36) and solvent system B (37) contained chloroform/methanol/pyridine/formic acid/water (20:25:15:1:2.5, v/v) and 6 g of boric acid. Radioactive phospholipids were visualized by PhosphorImaging analysis, and their relative densities were quantified using ImageQuant software.

For enzyme characterization studies, samples of the ³²P-labeled chloroform-soluble product of the reactions were dried and used directly for scintillation counting (35). All assays were conducted in triplicate with a standard deviation of ±5%. The enzyme assays were linear with time and protein concentration. A unit of PI 4-kinase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. Specific activity was defined as units per mg of protein. Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT enzyme kinetic model-fitting program (39).

Labeling and Analysis of Phosphoinositides-- Cells were labeled for 10 min with 50 µCi of [2-³H]inositol at the indicated temperatures as described by Wurmser and Emr (40) with the modifications of Audhya et al. (13). Extracts were prepared by lysis with glass beads in 4.5% perchloric acid. Phospholipids were deacylated with methylamine reagent, and the resulting glycerophosphoinositols were separated and analyzed by high performance liquid chromatography (41, 42).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of the S. cerevisiae LSB6 Gene Encoding PI 4-Kinase Activity-- Analysis of mammalian type II PI 4-kinase enzymes revealed a putative type II PI 4-kinase in S. cerevisiae, which is encoded by the LSB6 gene (8, 9). The LSB6 DNA sequence, which is found on chromosome X, does not have any sequence motifs that would suggest the existence of introns in the gene. The predicted protein product is 607 amino acids in length with a predicted minimum subunit molecular mass of 70.2 kDa. Analysis of the amino acid sequence using the TMpred program (www.ch.embnet.org/software/TMPRED_form.html) predicts that the protein has one presumptive transmembrane region. The MOTIF program (www.motif.genome.ad.jp) predicts that the LSB6 gene product has N-myristoylation and N-glycosylation sites and phosphorylation target sites for casein kinase II, protein kinase C, tyrosine kinase, and protein kinase A.

To examine the hypothesis that LSB6 encodes a PI 4-kinase enzyme, we examined the levels of PI 4-kinase activity in an lsb6 mutant and in cells that overexpress the LSB6 gene. The lsb6 mutant and mutant cells bearing the multicopy plasmid with the LSB6 gene were grown to exponential phase; cell extracts were prepared and assayed for PI 4-kinase activity. Analysis of the ³²P-labeled product of the reactions by thin layer chromatography using solvent system A showed that the PI 4-kinase activity in the mutant was reduced by 30% when compared with control cells (Fig. 1A). The remaining PI 4-kinase activity in the lsb6 mutant can be attributed to the PI 4-kinase activities encoded by the PIK1 (10) and STT4 (11) genes. The lsb6 mutant cells bearing the multicopy plasmid containing the LSB6 gene exhibited 10-fold greater PI 4-kinase activity when compared with the control (Fig. 1A). PI 4-kinase activity was similarly overexpressed in wild-type cells bearing the multicopy plasmid containing the LSB6 gene. To confirm that the product of the reaction was PI 4-phosphate, the chloroform-soluble ³²P-labeled product of the reaction catalyzed by the enzyme from cells overexpressing the LSB6 gene was subjected to thin layer chromatography using solvent system B. Solvent system B separates PI 4-phosphate from PI 3-phosphate based on the ability of PI 4-phosphate but not PI 3-phosphate to form a complex with boric acid (37). The product of the reaction comigrated with standard PI 4-phosphate (Fig. 1B).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   PI 4-kinase activity in S. cerevisiae wild-type cells, lsb6 mutant cells, and lsb6 cells overexpressing the LSB6 gene. A, cell extracts were prepared from the indicated cells and assayed for PI 4-kinase activity using 50 µg of protein. The 32P-labeled product of the reactions was extracted with chloroform and analyzed by thin layer chromatography using solvent system A. Reaction products were visualized by PhosphorImaging analysis and were quantified using ImageQuant software. The data shown are an average of two experiments. The break in the y axis is between 140 and 500%. B, a cell extract prepared from lsb6 mutant cells overexpressing the LSB6 gene was used to measure enzyme activity under the assay conditions for PI 3-kinase (lane 1) and for PI 4-kinase (lane 2). The 32P-labeled products of the reactions were analyzed by thin layer chromatography using solvent system B. Reaction products were visualized by PhosphorImaging analysis. The positions of PI 4-phosphate (PI4P) and PI 3-phosphate (PI3P) on a portion of a thin layer chromatogram are indicated in the figure. WT, SEY6210 [YEp352]; lsb6, AAY313 [YEp352]; lsb6/LSB6, AAY313 [pGH302].

Expression of LSB6-encoded PI 4-Kinase Activity in a Temperature-sensitive pik1^ts stt4^ts Double Mutant and Thermal Lability of the Enzyme-- We examined the PI 4-kinase activity in a temperature-sensitive pik1^ts stt4^ts double mutant. Cells were grown at 26 °C (permissive temperature); cell extracts were prepared, and PI 4-kinase activity was measured at 30 and at 37 °C. At 30 °C, the PI 4-kinase activity in the pik1^ts stt4^ts double mutant was reduced by 60% when compared with the wild-type control (Fig. 2A). Thus, at a semi-permissive temperature, the PI 4-kinase enzymes from the pik1^ts stt4^ts double mutant exhibited defects in enzyme activity. Introduction of the lsb6 mutation in the pik1^ts stt4^ts mutant resulted in an additional 20% decrease in PI 4-kinase activity (80% decrease overall). When PI 4-kinase activity was measured at 37 °C (restrictive temperature), there was a 90% reduction in activity in both the pik1^ts stt4^ts double mutant and in the pik1^ts stt4^ts lsb6 triple mutant (Fig. 2A). As a control, we assayed the PI 4-kinase activity from the lsb6 mutant at 30 and at 37 °C. As indicated above, the lsb6 mutation caused a 30% decrease in activity at 30 °C. Yet at 37 °C the PI 4-kinase in the lsb6 mutant was the same as that found in the wild-type control cells (Fig. 2A). In fact, the PI 4-kinase activity in the wild-type control cells was 30% lower at 37 °C when compared with the activity at 30 °C. The multicopy plasmid containing the LSB6 gene directed the overexpression of PI 4-kinase activity in the lsb6 mutant, the pik1^ts stt4^ts double mutant, and in the pik1^ts stt4^ts lsb6 triple mutant when the assays were performed at 30 °C (Fig. 2A). To our surprise, the levels of PI 4-kinase activity were not elevated in any of the mutants bearing the multicopy plasmid when the enzyme assays were conducted at 37 °C (Fig. 2A). These data indicated that the LSB6-encoded PI 4-kinase activity was temperature-sensitive.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Expression of LSB6-encoded PI 4-kinase activity in a temperature-sensitive pik1ts stt4ts double mutant and thermal lability of the enzyme. A, cell extracts (50 µg) from the indicated cells were assayed for PI 4-kinase activity at 30 and at 37 °C. The samples assayed at 37 °C were preincubated at 37 °C for 20 min. The 32P-labeled product of the reactions was extracted with chloroform and analyzed by thin layer chromatography using solvent system A. Reaction products were visualized by PhosphorImaging analysis and were quantified using ImageQuant software. The data shown are an average of two experiments. The break in the y axis is between 140 and 420%. B, samples (10 µg) of the membrane fraction from lsb6 mutant cells overexpressing the LSB6 gene were incubated at the indicated temperatures for 20 min. After the incubations, the samples were cooled on ice followed by the measurement of PI 4-kinase at 30 °C. WT, SEY6210 [YEp352]; lsb6, AAY313 [YEp352]; pik1ts stt4ts, AAY105 [pGH302]; pik1ts stt4ts lsb6, AAY317 [YEp352]; lsb6/LSB6, AAY313 [pGH302]; pik1ts stt4ts/LSB6, AAY105 [pGH302]; pik1ts stt4ts lsb6/LSB6, AAY317 [pGH302].

The effect of temperature on the stability of the LSB6-encoded PI 4-kinase activity was examined further. The membrane fraction derived from the lsb6 mutant overexpressing the LSB6 gene was incubated for 30 min at 20-70 °C in a temperature-controlled water bath. After incubation, the samples were cooled on ice to allow for protein renaturation, and PI 4-kinase activity was measured at 30 °C. Enzyme activity was unstable above 20 °C with total inactivation at 50 °C (Fig. 2B).

Heterologous Expression of the LSB6-encoded PI 4-Kinase in E. coli-- The overexpression of PI 4-kinase activity in cells bearing the multicopy plasmid containing the LSB6 gene suggested that LSB6 encoded a PI 4-kinase enzyme. However, this experiment did not rule out the possibility that the LSB6 gene was a regulatory gene whose product controlled the expression or activities of the PIK1-encoded and/or STT4-encoded PI 4-kinase enzymes. To test further the hypothesis that the LSB6 gene was the structural gene encoding a PI 4-kinase enzyme, we used heterologous expression of the gene in E. coli. This bacterium does not possess any type of phosphoinositide kinase (43). A His-tagged version of the LSB6 gene was cloned into an E. coli expression plasmid and then transformed into E. coli. The recombinant His-tagged protein was expressed in E. coli cells grown at 37 °C. However, the expressed protein did not exhibit PI 4-kinase activity. This was presumably because of the thermal lability of the enzyme. Thus, it was necessary to express the recombinant enzyme at 30 °C. Indeed, PI 4-kinase activity was present in the cell extract of cells grown at 30 °C. The His-tagged PI 4-kinase was highly purified from a cell extract by Ni-NTA-agarose affinity chromatography (Fig. 3A). SDS-PAGE analysis showed that the subunit molecular mass of the purified protein (78 kDa) was in good agreement with the predicted size of the His-tagged fusion protein. The purified His-tagged protein catalyzed the formation of PI 4-phosphate from PI and ATP in a dose-dependent manner (Fig. 3B). The specific activity of the recombinant was 0.3 nmol/min/mg. This activity was very low when compared with the specific activities (4-5 µmol/min/mg) of PI 4-kinase enzymes purified from yeast (6). The low level of activity exhibited by the recombinant enzyme may reflect incorrect folding or a difference in post-translational modification.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE of the LSB6 gene product purified from E. coli and demonstration that the LSB6-encoded enzyme is a PI 4-kinase. A, samples of molecular mass standards (Std), an E. coli cell extract, and the purified His-tagged LSB6-encoded PI 4-kinase were subjected to SDS-PAGE and stained with Coomassie Blue. The position of the recombinant LSB6-encoded PI 4-kinase (rLsb6p) is indicated. B, the PI 4-kinase activity of the purified recombinant LSB6-encoded enzyme was measured with the indicated protein concentrations.

LSB6 Is Not Essential but Overexpression of LSB6 Partially Suppresses the Lethal Phenotype of an stt4 Mutant-- Haploid lsb6 mutant cells were viable, exhibited no apparent gross morphological defects, and exhibited growth properties similar to wild-type cells when grown vegetatively in synthetic and in rich growth media at 30 °C. In addition, mating and sporulation were unaffected by the deletion of the LSB6 gene. At 30 °C, growth of lsb6 mutant cells was indistinguishable from wild-type cells using 2% glucose, 2% galactose, or 3% glycerol as the carbon source. Both lsb6 mutant cells and wild-type cells grew equally well at temperatures ranging from 17 to 37 °C, and on SC growth medium containing 1 M sorbitol or 1.5 M NaCl. Overall, these results indicated that the LSB6 gene was not essential for cell growth under common laboratory growth conditions and did not play a role in osmohomeostasis.

A multicopy plasmid bearing the LSB6 gene was transformed into pik1/PIK1 diploid and stt4/STT4 diploid cells. Sporulation and tetrad dissection analyses showed that the overexpression of the LSB6 gene did not suppress the lethal phenotypes of pik1 (10) and stt4 (44) mutants on YEPD growth medium. However, when the experiment was performed on YEPD plates supplemented with 1 M sorbitol, stt4 cells overexpressing the LSB6 gene formed small colonies after 6 days of growth (Fig. 4). The addition of sorbitol to the growth medium did not result in the growth of pik1 mutant cells containing the overexpressed LSB6 gene. The lsb6 mutation did not affect the growth phenotype of the pik1^ts mutant, the stt4^ts mutant, or the pik1^ts stt4^ts double mutant.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Overexpression of the LSB6 gene partially suppresses the lethal phenotype of an stt4 mutant. The multicopy plasmid pGH301 containing the LSB6 gene was transformed into stt4/STT4 diploid (AAY102) cells. Sporulation and tetrad dissection was performed on YEPD medium plates supplemented with 1 M sorbitol. Colony growth was scored after 6 days of incubation. The stt4 mutant cells bearing the plasmid containing the LSB6 gene are indicated by the white arrowheads.

The LSB6-encoded PI 4-Kinase Is a Membrane-associated Protein and Localizes to the Plasma Membrane and the Vacuole Membrane-- The association of PI 4-kinase activity with the total membrane and soluble fractions of the cell was examined. In wild-type cells, PI 4-kinase activity was associated with both the membrane and soluble fractions (Fig. 5A). Membrane-associated activity has been attributed to the STT4-encoded PI 4-kinase (44, 45), whereas the soluble-associated activity has been attributed to the PIK1-encoded PI 4-kinase (12). There was a 30% decrease in the PI 4-kinase activity associated with the membrane fraction of lsb6 mutant cells (Fig. 5A). The PI 4-kinase activity in the membrane fraction of lsb6 mutant cells bearing the LSB6 gene on the multicopy plasmid was 12-fold greater than the activity found in the membrane fraction derived from wild-type cells (Fig. 5A). In contrast, PI 4-kinase activity was not elevated in the soluble fraction from cells overexpressing the LSB6 gene (Fig. 5A). The association of the LSB6-encoded PI 4-kinase with the total membrane fraction was further examined by immunoblot analysis. For these studies, we utilized an overexpressed HA-tagged version of the enzyme. The HA-tagged PI 4-kinase was functional; it exhibited overexpressed levels of PI 4-kinase activity in the cell extract and membrane fraction similar to that of the untagged enzyme (Fig. 5A). Immunoblot analysis showed that HA-tagged PI 4-kinase (Lsb6p^HA), which migrated as a 75-kDa protein, was localized to the membrane fraction (Fig. 5B). These results indicated that the LSB6 gene product was a membrane-associated protein.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   PI 4-kinase activity in the membrane and soluble fractions of S. cerevisiae wild-type cells, lsb6 mutant cells, and lsb6 cells overexpressing the LSB6 gene. A, the total membrane (M) and soluble (S) fractions were isolated from the cell extract (E) of the indicated cells. Samples (50 µg) of the indicated fractions were used for the measurement of PI 4-kinase activity. The 32P-labeled product of the reactions was extracted with chloroform, analyzed by thin layer chromatography using solvent system A, and visualized by PhosphorImaging analysis. The portion of the thin layer chromatogram with the PI 4-phosphate reaction product is shown in the figure. B, samples (35 µg) of the indicated fractions from lsb6 mutant cells overexpressing the LSB6HA gene were subjected to immunoblot analysis using anti-HA antibodies (1 µg/ml). The positions of the HA-tagged LSB6-encoded PI 4-kinase protein (Lsb6pHA) and molecular mass standards are indicated in the figure. C, membranes from lsb6 mutant cells overexpressing the LSB6HA gene were suspended in buffer containing 1 M NaCl or with 1% Triton X-100 at a final protein concentration of 10 mg/ml. After incubation with shaking for 1 h at 5 °C, the suspensions were centrifuged at 100,000 × g for 1 h. The pellet (P) fraction from each treatment was suspended in the same volume as the supernatant (S) fraction, and equal volumes of the fractions were subjected to immunoblot analysis using anti-HA antibodies (1 µg/ml). A portion of the immunoblot is shown in the panel. WT, SEY6210 [YEp352]; lsb6, AAY313 [YEp352]; lsb6/LSB6, AAY313 [pGH302]; lsb6/LSB6HA, AAY313 [pGH303].

The membrane fraction was treated with 1 M NaCl and with 1% Triton X-100 to examine the nature of the association of the enzyme with the membrane. Following these treatments, the samples were separated into salt-extractable and detergent-extractable fractions. The PI 4-kinase protein was not released from the membrane after treatment with NaCl (Fig. 5C). On the other hand, treatment of the membrane with Triton X-100 resulted in the dissociation of the PI 4-kinase protein from the membrane (Fig. 5C). These properties were consistent with the conclusion that the LSB6-encoded PI 4-kinase is an integral membrane protein (46).

To characterize the localization of Lsb6p in living cells, the endogenous copy of the LSB6 gene was fused to GFP. By performing a pulse-chase labeling of Lsb6p-GFP with Tran³⁵S-label, we found that Lsb6p-GFP was expressed at very low levels before and after a 30-min chase with cold amino acids (Fig. 6A). Consistent with this observation, the endogenous level of Lsp6p-GFP was not detected by fluorescence microscopy. However, when overexpressed about 50-fold (Fig. 6A), Lsb6p-GFP was clearly visible on both the plasma membrane and the vacuolar membrane (Fig. 6B). Localization to the plasma membrane was consistent with our finding that overexpression of Lsb6p can partially suppress the lethal phenotype of stt4 mutant cells, because Stt4p has been localized previously to the plasma membrane (45).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Expression and fluorescent localization of Lsb6p-GFP. A, cells containing the LSB6-GFP fusion gene at the LSB6 locus (endo) and on a multicopy plasmid (overexp) were labeled with Tran35S-label to label Lsb6p followed by a 30-min chase with cold methionine and cysteine. The Lsp6p-GFP fusion protein was then immunoprecipitated from cell extracts with anti-GFP antibodies and subjected to SDS-PAGE and fluorography. The position of the Lsp6p-GFP fusion protein is indicated in the figure. B, cells overexpressing the LSB6-GFP fusion gene were labeled with CMAC, a vital dye that stains the vacuole lumen. The Lsb6p-GFP fusion protein and the vacuole lumen were visualized by fluorescence microscopy. Cells were also observed by Nomarski optics. Observations were based on the examination of at least 150 cells.

Enzymological Properties of the LSB6-encoded PI 4-Kinase Activity-- The assay conditions for the different PI 4-kinase enzymes described from S. cerevisiae differ (11, 12, 14, 16). Therefore, it was important to determine the basic enzymological properties of the LSB6-encoded PI 4-kinase so that the activity of this enzyme could be measured under optimal assay conditions. The recombinant enzyme expressed in E. coli was not used in our studies to examine the enzymological properties of the enzyme. The specific activity of the recombinant enzyme was low, and we were concerned that the enzyme was not subject to post-translational modifications (e.g. myristoylation, glycosylation, and phosphorylation) that may be required for optimal activity. Accordingly, membranes derived from lsb6 cells overexpressing the LSB6 gene were used as the source of enzyme for these studies. PI 4-kinase was measured with a Tris-maleate-glycine buffer at pH values ranging from 5 to 10. Optimum activity was found between pH 6.5 and 7.5 (Fig. 7A). The effect of Triton X-100 on PI 4-kinase activity is shown in Fig. 7B. The addition of 3.2 mM Triton X-100 to the assay mixture resulted in a 40-fold stimulation of PI 4-kinase activity. The apparent inhibition of activity at Triton X-100 concentrations above 3.2 mM was characteristic of surface dilution kinetics (47). PI 4-kinase activity exhibited a dose-dependent requirement for magnesium ions with maximum activity at a final concentration of 10 mM (Fig. 7C). The magnesium ion requirement could be partially substituted by manganese ions (Fig. 7D).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of pH, Triton X-100, magnesium, and manganese on LSB6-encoded PI 4-kinase activity. Membranes (10 µg) prepared from lsb6 mutant cells overexpressing the LSB6 gene were assayed for PI 4-kinase activity at the indicated pH values with 50 mM Tris maleate-glycine buffer (A), the indicated concentrations of Triton X-100 (B), the indicated concentrations of MgC12 (C), and the indicated concentrations of MnC12 (D).

The function of Triton X-100 in the assay for many lipid-dependent enzymes is to form a mixed micelle with the lipid substrate providing a surface for catalysis (47). Because the LSB6-encoded PI 4-kinase exhibited surface dilution kinetics, the kinetic analysis of the enzyme was performed using Triton X-100/PI-mixed micelles. Accordingly, the concentration of PI in the mixed micelles was expressed as a surface concentration in mol % as opposed to a molar concentration (47). In these experiments, the enzyme was measured such that PI 4-kinase activity was only dependent on the surface concentration of PI (i.e. at a molar PI concentration of 0.2 mM) and independent on the molar concentration of PI (15, 16, 47). Kinetic experiments were also performed using a saturating concentration of magnesium ions (10 mM). Under these conditions, ATP in the assay system existed in the form of Mg²⁺-ATP complexes (48). The enzyme exhibited positive cooperative kinetics with respect to the surface concentration of PI at a set ATP concentration of 2.5 mM (Fig. 8A). Analysis of the kinetic data according to the Hill equation yielded a Hill number of 1.9 and an apparent K_m value for PI of 3 mol %. PI 4-kinase activity followed typical saturation kinetics with respect to ATP at a set PI surface concentration of 6 mol % (Fig. 8B). Analysis of the data according to the Michaelis-Menten equation yielded an apparent K_m value for ATP of 0.65 mM.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Dependence of LSB6-encoded PI 4-kinase activity on the surface concentration of PI and on ATP. A, PI 4-kinase activity was measured as a function of the surface concentration (mol %) of PI at a set ATP concentration of 2.5 mM. The molar concentration of PI was held constant at 0.2 mM, whereas the Triton X-100 concentration was varied. B, PI 4-kinase activity was measured as a function of the molar concentration of ATP at a set PI surface concentration of 6 mol %. Membranes (10 µg) prepared from lsb6 mutant cells overexpressing the LSB6 gene were used for these experiments.

The LSB6-encoded enzyme was examined for its ability to utilize other phosphoinositide molecules as substrates. Under standard assay conditions, the enzyme did not catalyze the incorporation of the -phosphate of ATP into 0.2 mM lyso-PI, PI 3-phosphate, PI 4-phosphate, PI 5-phosphate, and PI 4,5-bisphosphate. In addition, the enzyme did not phosphorylate diacylglycerol and phosphatidate. The addition of 2.5 mM inositol and inositol 1-phosphate did not inhibit PI 4-kinase activity indicating that these water-soluble molecules did not serve as substrates for the enzyme.

Effect of the LSB6 Gene on the Cellular Levels of Phosphoinositides-- lsb6 mutant cells and wild-type cells bearing the multicopy plasmid with the LSB6 gene were grown to the exponential phase of growth. Cells were then incubated with [2-³H]inositol for 10 min at 26 °C to label the total cellular pool of phosphoinositides. Phospholipids were extracted from the cells and deacylated with methylamine reagent, and the resulting glycerophosphoinositols were analyzed by high performance liquid chromatography. This analysis showed that the synthesis of PI 4-phosphate and PI 4,5-bisphosphate in lsb6 mutant cells and in wild-type cells that overexpressed the LSB6 gene was not significantly different from that found in wild-type cells (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PIK1-encoded (10) and STT4-encoded (11) PI 4-kinase enzymes of S. cerevisiae contain a characteristic catalytic kinase domain (3, 4, 49) that is also found in the VPS34-encoded PI 3-kinase (41, 50). Additional genes that encode PI kinase enzymes with this domain do not exist in the yeast data base. This information, along with the fact that shifting pik1^ts stt4^ts double mutant cells to the restrictive temperature halts the synthesis of PI 4-phosphate and PI 4,5-bisphosphate (13), has led to the conclusion that PIK1 and STT4 may be the only genes encoding PI 4-kinase enzymes in S. cerevisiae (7, 13, 51, 52). In this work, we examined the hypothesis that the S. cerevisiae LSB6 gene encodes a PI 4-kinase because its deduced protein sequence shows similarity with mammalian type II PI 4-kinase enzymes (8, 9).

Deletion of the LSB6 gene resulted in a decrease in PI 4-kinase activity in cell extracts. A multicopy plasmid containing the LSB6 gene directed the overexpression of PI 4-kinase activity in cell extracts of wild-type cells, in an lsb6 mutant, in a pik1^ts stt4^ts double mutant, and in a pik1^ts stt4^ts lsb6 triple mutant. Moreover, the heterologous expression of the LSB6 gene in E. coli resulted in the expression of a protein that possessed PI 4-kinase activity. Collectively, these data provided strong evidence for the identification of the LSB6 gene as the structural gene encoding a PI 4-kinase activity in S. cerevisiae. The LSB6-encoded PI 4-kinase was localized to the membrane fraction of the cell and, when overexpressed, was observed on both the plasma membrane and the vacuolar membrane. The enzyme behaved like an integral membrane protein (46); it could be dissociated from membranes with 1% Triton X-100 but could not be dissociated from membranes with 1 M NaCl. Additional studies will be required to address the nature of this association (e.g. protein-lipid and protein-protein interactions).

Analysis of PI 4-kinase activity at 30 and at 37 °C in cells overexpressing the LSB6 gene, and temperature inactivation studies of the enzyme in the membrane fraction showed that the LSB6-encoded PI 4-kinase was temperature-sensitive in vitro. This property provides a logical explanation for why phosphoinositide synthesis stops in the pik1^ts stt4^ts double mutant after shifting cells to 38 °C (13).

Construction of the lsb6 mutant revealed that the LSB6 gene was not essential for cell growth in S. cerevisiae. The mutant lacked an identifiable phenotype. Vegetative growth and cell morphology of the lsb6 mutant were indistinguishable from wild-type cells. Deletion of the LSB6 gene did not affect the synthesis of the total phosphoinositide pool in vivo. This observation may be explained by the fact that the LSB6 gene was not highly expressed in wild-type cells and that the LSB6-encoded PI 4-kinase may provide only a small pool of PI 4-phosphate at the plasma membrane and/or the vacuolar membrane. Moreover, the overexpression of the LSB6 gene in wild-type cells did not affect the synthesis of phosphoinositides. The overexpression of the STT4 gene similarly does not affect phosphoinositide synthesis in vivo,² whereas overexpression of the PIK1 gene results in only a modest increase (1.6-fold) in phosphoinositide synthesis.² These data suggest that the activities of the PI 4-kinase enzymes must be highly regulated to control the levels of phosphoinositides in vivo.

Unlike the LSB6 gene, the PIK1 (10) and STT4 (44) genes are essential for growth of S. cerevisiae. The PIK1-encoded and STT4-encoded PI 4-kinase enzymes synthesize discrete pools of PI 4-phosphate that play essential roles in cell physiology (13). The PIK1-encoded activity is required for protein secretion and endocytosis (13, 53, 54). The STT4-encoded activity is an essential component of the PKC1 pathway (11, 45), is required for maintaining vacuole morphology and actin cytoskeleton organization (13), and plays a role in the regulation of intracellular aminophospholipid transport (55). The overexpression of the LSB6 gene did not suppress the lethal phenotype of the pik1 mutant, indicating that LSB6 and PIK1 do not have overlapping functions in cell physiology. However, overexpression of the LSB6 gene could partially suppress the lethal phenotype of stt4 mutant cells. This observation provided evidence that Lsb6p functions as a PI 4-kinase in vivo. Previous studies (45) have shown that Stt4p localizes to the plasma membrane and generates an essential pool of PI 4-phosphate required for normal activity of the PKC1 pathway. When overexpressed, Lsb6p also localizes to the plasma membrane and likely suppresses the lethal phenotype of stt4 mutant cells by producing PI 4-phosphate at this cellular location. However, stt4 mutant cells overexpressing Lsb6p grew extremely poorly and only in the presence of sorbitol. This indicates that Lsb6p cannot fully complement the essential roles that Stt4p plays at the plasma membrane. Surprisingly, the lsb6 mutation did not reveal any synthetic growth defects/phenotypes in the stt4^ts mutant, suggesting that the LSB6-encoded PI 4-kinase does not play a major role in STT4-mediated PI 4-phosphate production at the plasma membrane, perhaps due to its very low expression levels in vegetatively growing cells.

Type II-like 55-kDa and 45-kDa PI 4-kinase enzymes have been solubilized from membranes with Triton X-100 and purified to near-homogeneity from S. cerevisiae (14-16). The enzymological properties of these two enzymes differ from each other with respect to pH optima, cofactor dependence, Triton X-100 dependence, binding, and catalytic properties for the substrate PI and regulation by nucleotides and by phospholipids (6, 14-18). Due to the lack of protein sequence information from the 55- and 45-kDa PI 4-kinase enzymes, it has been speculated that they may represent proteolytic fragments of the soluble 125-kDa type III PIK1-encoded PI 4-kinase enzyme (10). However, the membrane-associated nature of the 55- and 45-kDa enzymes indicates that these enzymes would differ from the PIK1-encoded PI 4-kinase. Analysis of the basic enzymological properties of the LSB6-encoded PI 4-kinase activity indicated that this enzyme was more similar to the 55-kDa PI 4-kinase when compared with the 45-kDa enzyme (Table III). The K_m values for PI of the LSB6-encoded PI 4-kinase and the 55-kDa PI 4-kinase were similar, and the magnesium ion requirement for both enzymes could be substituted by manganese ions. In addition, the pH optimum for the LSB6-encoded PI 4-kinase and 55-kDa PI 4-kinase reactions were the same. These biochemical similarities raised the suggestion that the LSB6 gene may encode the 55-kDa PI 4-kinase. The differences in the molecular masses of the LSB6-encoded PI 4-kinase and the 55-kDa PI 4-kinase may be explained by proteolysis of the LSB6-encoded protein. The 55-kDa PI 4-kinase is very labile during the eight-step purification scheme used to purify the enzyme (16, 56). An alternative explanation is that the 55-kDa enzyme is encoded by another gene. Additional studies are needed to clarify the relationship of the 55- and 45-kDa PI 4-kinases with other PI 4-kinase enzymes in yeast.

The conservation of a type II PI 4-kinase enzyme from yeast to human cells (8, 9) suggests that the LSB6-encoded enzyme may play an important physiological role in yeast. The LSB6 gene was originally identified in a two-hybrid screen using the Las17p/Bee1p protein as bait (19). Lsp17/Bee1p is a protein component of actin patches and plays a role in actin patch assembly, budding, and cytokinesis (20). In a global two-hybrid analysis, Lsb6p was also shown to interact Atp14p and Ism1p (57). Atp14p is component h of the mitochondrial proton-transporting ATP synthase complex (58), and Ism1p is a mitochondrial isoleucyl-tRNA ligase (59). Additional studies will be required to determine the significance of the interactions of Lsb6p with Lsp17p/Bee1p, Atp14p, and Ism1p. Because the deletion of LSB6 failed to exhibit any striking phenotypes or affect total phosphoinositide levels in vivo, our results are consistent with previous findings that under common laboratory conditions, the PIK1 and STT4 genes account for the major PI 4-kinase activities in S. cerevisiae (13). Further studies will be required to determine what specialized cellular role(s) the LSB6-encoded PI 4-kinase may play.

	ACKNOWLEDGEMENTS

We thank Avula Sreenivas for helpful suggestions during the course of this work. We also thank Charles Zuker for generously providing anti-GFP antibodies.

	FOOTNOTES

^* This work was supported in part by United States Public Health Service Grants GM-28140 (to G. M. C.) and CA-58689 (to S. D. E.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ An investigator of the Howard Hughes Medical Institute.

To whom correspondence and reprint requests should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 908-932-9611 (ext. 217); Fax: 908-932-6776; E-mail: carman@aesop.rutgers.edu.

Published, JBC Papers in Press, October 1, 2002, DOI 10.1074/jbc.M207996200

² J. Audhya and S. D. Emr, unpublished data.

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES