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• W2080209900 abstract "Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323–1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP6) to diphosphoinositol pentakisphosphate, a “high energy” candidate regulator of cellular trafficking. We have now studied the significance of InsP6 kinase activity inSaccharomyces cerevisiae by disrupting the kinase gene. These ip6kΔ cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60–80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP6kinases were identified; inositol pentakisphosphate (InsP5) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP4), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP5(K m = 1.2 μm) and InsP6(K m = 6.7 μm) compete for phosphorylation in vivo. This is the first time a PP-InsP4 synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP6. Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged. Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323–1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP6) to diphosphoinositol pentakisphosphate, a “high energy” candidate regulator of cellular trafficking. We have now studied the significance of InsP6 kinase activity inSaccharomyces cerevisiae by disrupting the kinase gene. These ip6kΔ cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60–80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP6kinases were identified; inositol pentakisphosphate (InsP5) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP4), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP5(K m = 1.2 μm) and InsP6(K m = 6.7 μm) compete for phosphorylation in vivo. This is the first time a PP-InsP4 synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP6. Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged. diphosphoinositol tetrakisphosphate diphosphoinositol pentakisphosphate bis-diphosphoinositol trisphosphate bis-diphosphoinositol tetrakisphosphate 5- (and 6-)carboxy-2′,7′-dichlorofluorescein diacetate 5- (and 6-)carboxy-2′,7′-dichlorofluorescein human diphosphoinositol polyphosphate phosphohydrolase 4,5,6)P4,d-myo-inositol 3,4,5,6-tetrakisphosphate inositol hexakisphosphate inositol pentakisphosphate 3,4,5,6)P5,d-myo-inositol 1,3,4,5,6-pentakisphosphate high pressure liquid chromatography 4-morpholineethanesulfonic acid The very dynamic turnover of the “high energy” diphosphorylated inositol polyphosphates (PP-InsP4, PP-InsP5, and [PP]2-InsP4)1may represent a molecular switching activity that regulates intracellular trafficking (see Ref. 1Safrany S.T. Caffrey J.J. Yang X. Shears S.B. Biol. Chem. 1999; 380: 945-951Crossref PubMed Scopus (33) Google Scholar for a review). For example, PP-InsP5 represents the most potent known inhibitor of AP180-mediated assembly of clathrin cages, a key step in the endocytic retrieval of discharged synaptosomal vesicles (2Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Other proteins that participate in intracellular trafficking can bind PP-InsP5 very tightly, including coatomer (3Fleischer B. Xie J. Mayrleitner M. Shears S.B. Palmer D.J. Fleischer S. J. Biol. Chem. 1994; 269: 17826-17832Abstract Full Text PDF PubMed Google Scholar, 4Ali N. Duden R. Bembenek M.E. Shears S.B. Biochem. J. 1995; 310: 279-284Crossref PubMed Scopus (47) Google Scholar) and AP2 (5Shears S.B. Ali N. Craxton A. Bembenek M.E. J. Biol. Chem. 1995; 270: 10489-10497Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The high affinity with which PP-InsP5 binds to myelin proteolipid protein may be important for the vesicular delivery of the latter to the myelin sheath (6Yamaguchi Y. Ikenaka K. Niinobe M. Yamada H. Mikoshiba K. J. Biol. Chem. 1996; 271: 27838-27846Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Prior experiments with intact cells have shown that Ins(1,3,4,5,6)P5 and InsP6 serve as metabolic stockpiles for the formation of the diphosphorylated inositol polyphosphates (7Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar, 8Stephens L.R. Radenberg T. Thiel U. Vogel G. Khoo K.-H. Dell A. Jackson T.R. Hawkins P.T. Mayr G.W. J. Biol. Chem. 1993; 268: 4009-4015Abstract Full Text PDF PubMed Google Scholar). InsP6 is the precursor for PP-InsP5, which is further phosphorylated to [PP]2-InsP4 (5Shears S.B. Ali N. Craxton A. Bembenek M.E. J. Biol. Chem. 1995; 270: 10489-10497Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar, 8Stephens L.R. Radenberg T. Thiel U. Vogel G. Khoo K.-H. Dell A. Jackson T.R. Hawkins P.T. Mayr G.W. J. Biol. Chem. 1993; 268: 4009-4015Abstract Full Text PDF PubMed Google Scholar, 9Huang C.-F. Voglmaier S.M. Bembenek M.E. Saiardi A. Snyder S.H. Biochemistry. 1998; 37: 14998-15004Crossref PubMed Scopus (43) Google Scholar, 10Voglmaier S.M. Bembenek M.E. Kaplin A.I. Dormán G. Olszewski J.D. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4305-4310Crossref PubMed Scopus (132) Google Scholar). These reactions appear to take place within a metabolic pool that is separate from that in which Ins(1,3,4,5,6)P5 and PP-InsP4 are interconverted (7Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar). Thus, there are two metabolic pools of inositol diphosphates that are turned over in parallel cycles. There are increasing efforts to characterize the activities of the enzymes that regulate the turnover of PP-InsP4, PP-InsP5, and [PP]2-InsP4. Several phosphatases (diphosphoinositol polyphosphate phosphohydrolases) that hydrolyze these compounds have been described (11Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar). Two forms of InsP6 kinase (types 1 and 2), derived from distinct genes, have been cloned from mammals (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 14Schell M.J. Letcher A.J. Brearley C.A. Biber J. Murer H. Irvine R.F. FEBS Lett. 1999; 461: 169-172Crossref PubMed Scopus (74) Google Scholar). At least in mammals, the further phosphorylation of PP-InsP5to [PP]2-InsP4 appears to be the function of a separate enzyme that has been purified from rat brain (9Huang C.-F. Voglmaier S.M. Bembenek M.E. Saiardi A. Snyder S.H. Biochemistry. 1998; 37: 14998-15004Crossref PubMed Scopus (43) Google Scholar) but not yet cloned. A yeast InsP6 kinase has also been cloned that shows approximately 30% sequence similarity to the two mammalian InsP6 kinases (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). We also recently reported that PP-InsP5 and [PP]2-InsP4 are present in Saccharomyces cerevisiae (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar). A notable omission from our understanding of the intricacies of the turnover of higher inositol phosphates is any characterization of the enzyme(s) that phosphorylate Ins(1,3,4,5,6)P5 to PP-InsP4. Thus, an important goal of this study was to identify this “missing link” in inositide research: the PP-InsP4 synthase. We now report that PP-InsP4in mammals is synthesized by the type 1 and type 2 forms of the InsP6 kinase. In addition, we show that these enzymes can further phosphorylate PP-InsP4, thereby forming a hitherto unknown inositol polyphosphate. We have further investigated the activity of the yeast enzyme in several ways. We have characterized its substrate specificity in vitro. In this study, we demonstrate that disrupting the InsP6 kinase gene of S. cerevisiae(ip6kΔ) influences inositol polyphosphate levels in a unique fashion. We also show that ip6kΔ yeast cells have fragmented vacuoles. These data support an important role for diphosphoinositol polyphosphates in regulating intracellular trafficking. The various InsP6 kinases used in this study were incubated for various times at 37 °C in 25 or 50 μl of buffer containing 20 mm HEPES, pH 7.0, with KOH, 12 mm MgSO4, 1 mm dithiothreitol, 10 mm ATP, 20 mm phosphocreatine, 1 mmEDTA, 0.02 mg/ml phosphocreatine kinase (Calbiochem 238395) and 0.5 mg/ml bovine serum albumin. The appropriate 3H-labeled inositol phosphate was added as indicated. Assays were quenched with ice-cold perchloric acid and neutralized as described previously (16Shears S.B. Shears S.B. Signalling by Inositides: A Practical Approach. Oxford University Press, Oxford, UK1997: 33-52Google Scholar). Where specifically indicated, reactions were quenched by incubation for 3 min at 100 °C. Control experiments showed that all kinase activity was inactivated by this heat treatment, but none of the diphosphorylated inositol phosphates were degraded. The HPLC analysis of [3H]inositol-labeled yeast cells was performed as described previously (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar). Assays of the activities of recombinant enzymes employed HPLC using a Partisphere SAX column (Krackler Scientific, Durham, NC) that was eluted with a gradient generated by mixing Buffer A (1 mm Na2EDTA) and Buffer B (Buffer A plus 1.3 m(NH4)2HPO4, pH 3.85, with H3PO4) as follows: 0–5 min, 0% B; 5–10 min, 0–45% B; 10–60 min, 45–100% B; 60–70 min, 100% B. 1-ml fractions were collected. In some experiments, particularly when a new HPLC column was installed, the percentage of B at 10 min was increased to 50%. This change in the gradient, plus the tendency of inositol phosphates to elute earlier as the column aged, means that slightly different elution properties are seen in the different figures shown in this study. Recombinant hDIPP2α was prepared as described previously (11Caffrey J.J. Safrany S.T. Yang X. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The cDNAs for mammalian InsP6 kinase types 1 and 2 were recovered from the pCMV-glutathione S-transferase vector used previously (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The type 1 kinase cDNA was amplified using the following primers: 5′-GCACTCGAGAATGTGTGTTTGTCAAAC-3′ and 5′-GCTAAGCTTAGGGCCTACTGGTTCTC-3′; the polymerase chain reaction product was subcloned into the XhoI and HindIII sites of the pTrcHisB expression vector (Invitrogen). The cDNA for the type 2 kinase was amplified using the following primers: 5′-CGACTCGAGGATGAGCCCAGCCTTCAG-3′ and 5′-GCATTCGAAC TCACTCCCCACTCTCCTC-3; the polymerase chain reaction product was subcloned into the XhoI and BstBI restriction sites of pTrcHisB. The methods used to transform Escherichia coli (strain BL21), to induce with isopropyl-1-thio-β-d-galactopyranoside, and to isolate the poly(His)-tagged proteins using Talon resin (CLONTECH), were all according to the manufacturer's recommendations. The recombinant yeast InsP6 kinase was produced as described previously (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The YDR017C open reading frame in S. cerevisiae comprises thekcs1 gene that encodes the yeast InsP6 kinase. Using strain PJ69–2A of S. cerevisiae, the portion of thekcs1 gene that encodes the C terminus of the kcs1protein from amino acid 575 to the C-terminal stop codon was replaced by the dominant kanr marker gene using the KanMX4 expression construct as described previously (17Güldener U. Heck S. Fiedler T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1372) Google Scholar). The oligonucleotides used for the gene disruption were: 5′-GAAGGAAAAGAAACTCTAATACGACTACAATGGGAAACCATAATGCATAGGCCACTAGTGGATCTG-3′ and 5′-TAAGCGCAGCTAAAAGAATATTCATTAGTTCTATCCTTTCTTTTCAGCTGAAGCTTCGTACGC-3′. 10 μg of total RNA prepared from exponentially growing yeast was fractionated on a 1% agarose/MOPS-formaldehyde gel and transferred to Hybond N+ membranes, according to the manufacturer's instructions (Amersham Pharmacia Biotech). The blot was hybridized with a 1.2-kilobaseBamHI-NotI fragment corresponding to the 3′-region of the gene. This fragment was obtained by the digestion of the cDNA for the yeast InsP6 kinase (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Wild-type and ip6kΔ yeast were grown at 30 °C in YPD medium (20 g/liter peptone, 10 g/liter yeast extract, 2% glucose). Yeasts from 5 ml of early logarithmic phase cultures (A 600 = 0.4–0.6) were collected and resuspended in 100 μl of medium containing 50 mm sodium citrate buffer, pH 5.0, 2% glucose, 10 μm carboxy-DCFDA. Cells were incubated at room temperature for 20 min. Fluorescence (excitation = 504 nm, emission = 529 nm) was visualized using a Zeiss Axioskop microscope with 100× objective. Nonradioactive InsP6 and Ins(1,3,4,5,6)P5 were purchased from Calbiochem (La Jolla, CA) and the Alexis Corporation (San Diego, CA), respectively. Stock solutions were prepared in 1 mm EDTA. Carboxy-DCFDA was purchased from Molecular Probes, Inc. (Eugene, OR). [3H]InsP6 was purchased from NEN Life Science Products. [3H]Ins(1,3,4,5,6)P5 and [3H]Ins(3,4,5,6)P4 were isolated from 5-day-old [3H]inositol-labeled chick erythrocytes (18Stephens L.R. Downes C.P. Biochem. J. 1990; 265: 435-452Crossref PubMed Scopus (53) Google Scholar). [PP]2-[3H]InsP4 was obtained by phosphorylation of [3H]InsP6 using partly purified enzyme preparations from rat brain (19Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). PP-[3H]InsP5 and PP-[3H]InsP4 were prepared by phosphorylation of [3H]InsP6 and [3H]Ins(1,3,4,5,6)P5 respectively, using the type 1 InsP6 kinase (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). 14C-Labeled Ins(1,3,4,5,6)P5 was isolated from [14C]inositol-labeled, parotid acinar glands (20Hughes P.J. Hughes A.R. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1989; 264: 19871-19878Abstract Full Text PDF PubMed Google Scholar). All of the radiolabeled inositol phosphates that we synthesized were purified by HPLC and desalted (7Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar). We recently showed that PP-InsP5 and [PP]2-InsP4 were present in S. cerevisiae (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar). One goal of this study was to determine the importance of the yeast InsP6 kinase to the synthesis of the diphosphorylated inositol phosphates in vivo. The yeast InsP6 kinase protein is about three times larger in mass than either of the type 1 and type 2 forms of the mammalian InsP6 kinases (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The domain of the yeast InsP6 kinase that is homologous to the smaller, mammalian enzymes resides in a region close to the C terminus. The latter portion of the yeast protein was deleted by disrupting the appropriate 3′-region of the kinase gene (Fig. 1 and see “Experimental Procedures”). To confirm the correct integration of the marker gene, two diagnostic Southern blots were performed. Yeast genomic DNA was digested by SnaBI, and a band of approximately 5.5 kilobases was detected only in the gene-disrupted strain upon hybridization with the kanr gene (data not shown). Coincidentally, the kanrcassette used for the gene disruption is almost identical in size to the InsP6 kinase gene it replaces (Fig. 1). Thus, a band of approximately 5.5 kilobases was detected only in wild-type cells upon hybridization with a probe corresponding to the deleted region of the InsP6 kinase gene (data not shown). The success of the gene disruption was also verified by Northern analysis of wild-type and mutant yeast (Fig. 1). The gene-disrupted strain is hereafter designated ip6kΔ. We investigated the consequences of the gene disruption upon the inositol phosphate profile of wild-type and ip6kΔ cells. Levels of PP-InsP5 in ip6kΔ cells were >80% lower than those of wild-type cells (TableI). This result demonstrates that this kinase is quantitatively important for the expression of PP-InsP5 synthase activity in vivo. Nevertheless, our results indicate that there must be an alternative, hitherto unrecognized, pathway for PP-InsP5 synthesis in yeast. Levels of [PP]2-InsP4 were 60% lower in the ip6kΔ cells compared with the wild type (Table I). This result indicates that the InsP6 kinase plays only a partial role in the pathway of [PP]2-InsP4synthesis. Note also that the levels of Ins(1,3,4,5,6)P5 in the ip6kΔ cells were 80% lower than those of wild-type cells (Table I). Thus, in yeast, there is an unexpected link between the metabolism of PP-InsP5 and Ins(1,3,4,5,6)P5.Table IComparison of levels of higher inositol phosphates in wild-type and ip6kΔ strains of S. cerevisiaeInositol phosphateWT ([3H] dpm)ip6kΔ ([3H] dpm)ip6kΔ/WTIns(1,3,4,5,6)P53238 ± 1274718 ± 1620.22Ins(1,2,4,5,6)P510,624 ± 306610,287 ± 32490.97InsP6169,333 ± 18,883133,720 ± 24,9310.79PP-InsP5951 ± 120161 ± 300.17[PP]2-InsP41505 ± 199613 ± 920.41Levels of inositol phosphates were determined by HPLC as described under “Experimental Procedures.” Data are the means and S.E. from three experiments. Levels of the less phosphorylated inositol phosphates (InsPn, where n = 1–4) were similar in both wild-type (WT) and ip6kΔ cells. Open table in a new tab Levels of inositol phosphates were determined by HPLC as described under “Experimental Procedures.” Data are the means and S.E. from three experiments. Levels of the less phosphorylated inositol phosphates (InsPn, where n = 1–4) were similar in both wild-type (WT) and ip6kΔ cells. The ip6kΔ cells grew more slowly than the wild-type cells at 23 and 30 °C (Fig. 2). At 37 °C, the ip6kΔ cells did not grow at all (Fig. 2). Prior to the identification of the yeast InsP6 kinase (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), the gene that encodes this protein was known as kcs1 (21Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar). In that earlier study, the deletion of the kcs1/InsP6kinase gene from S. cerevisiae yielded no apparent growth phenotype (21Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar), possibly because that strain of yeast had a different genetic background from the strain that we have used. We (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar) and others (22York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar) have previously shown that drastic impairment of the pathway of InsP6 synthesis in S. cerevisiaeis accompanied by a decreased efficiency in the rate of export of mRNA from the nucleus. We (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar) did point out, however, that mRNA export may be regulated by metabolites of InsP6(such as PP-InsP5 and [PP]2-InsP4) rather than by InsP6itself. Indeed, we previously showed that one particular gene-disrupted strain of yeast (ipmkΔ), which displayed an impaired mRNA export phenotype and decreased synthesis of InsP6, also had 60–80% lower levels of PP-InsP5 and [PP]2-InsP4 (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar). The ip6kΔ cells gave us the first opportunity to study whether mRNA export was affected by quantitatively similar changes in levels of PP-InsP5 and [PP]2-InsP4, under conditions where InsP6 levels were not significantly affected (Table I). We measured mRNA export as described previously (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar) but found no significant difference between wild-type andip6kΔ cells (data not shown). This negative result redirected our efforts to the hypothesis (see the Introduction) that diphosphoinositol polyphosphates regulate protein trafficking. In yeast cells, several different vesicle transport pathways converge upon the vacuole (23Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar). This organelle receives endocytic traffic from the cell surface as well as biosynthetic traffic from the Golgi apparatus (23Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar, 24Conibear E. Stevens T.H. Cell. 1995; 83: 513-516Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Thus, we studied the effect of the deletion of the InsP6kinase upon vacuole morphology. The luminal interior of yeast vacuoles was identified by incubating cells with membrane-permeable carboxy-DCFDA (25Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods. Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). Once the probe has entered the vacuoles, the acetate groups are hydrolyzed by nonspecific esterases, forming the less membrane permeable and fluorescent carboxy-DCF (25Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods. Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). Fluorescence detection of the vacuolar space in wild-type cells revealed the usual complement (26Srivastava A. Jones E.W. Genetics. 1998; 148: 85-98Crossref PubMed Google Scholar) of one large vacuole (Fig.3). In contrast, the ip6kΔ strain contained several smaller, fragmented vacuoles (Fig. 3). This altered vacuolar morphology may reflect defects in the fusion into vacuoles of small vesicles derived from endocytosis or from the trans-Golgi network. The intensity of staining is also decreased in the null strain (Fig. 3), possibly because of less active intraluminal esterase activity in the ip6kΔ cells. Could the lower levels of [PP]2-InsP4 in the ip6kΔ yeast cells (Table I) reflect an unexpected ability of the yeast InsP6 kinase to phosphorylate PP-InsP5? To examine this question, we compared the rates of phosphorylation of both InsP6 and PP-InsP5 by the recombinant yeast InsP6 kinase. Trace amounts of [3H]InsP6 were almost completely phosphorylated to PP-[3H]InsP5 (Fig.4 A and Ref. 13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The yeast kinase was also separately incubated with PP-[3H]InsP5, but only approximately 5% of the substrate was converted to [PP]2-InsP4(Fig. 4 C), and in any case, even this slow reaction required 5-fold higher concentrations of enzyme than were used for the assays of InsP6 phosphorylation (Fig. 4 A). Furthermore, levels of InsP6 in S. cerevisiae are 180-fold higher than those of PP-InsP5 (Table I). These data indicate that the yeast InsP6 kinase does not provide an efficient route for [PP]2-InsP4 synthesisin vivo. We must therefore search for a separate yeast PP-InsP5 kinase with greater catalytic efficiency that can account for [PP]2-InsP4 synthesis in vivo. We also found that the yeast InsP6 kinase phosphorylated Ins(1,3,4,5,6)P5 (Fig. 4 B). TheV max values for InsP5 and InsP6 were each approximately 2 μmol/mg/min. The affinity of the enzyme for InsP6 (mean K m = 3.3 μm) was about 3-fold less than the affinity for InsP5 (mean K m = 1.2 μm). This is the first time an enzyme with PP-InsP4 synthase activity has been identified. In the case of S. cerevisiae, cellular levels of InsP6 are 50-fold higher than those of InsP5 (Table I), so we would not anticipate substantial phosphorylation of InsP5 by this kinase in intact yeast cells. However, in mammalian cells, levels of Ins(1,3,4,5,6)P5 and InsP6 are very similar to each other (27Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (153) Google Scholar). We therefore next investigated whether either of the two mammalian InsP6 kinases (named types 1 and 2; see Ref.13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) could also phosphorylate Ins(1,3,4,5,6)P5. The type 1 InsP6 kinase was expressed in E. coli as a His-tagged protein and purified using Talon resin (Fig. 5). The enzyme was incubated with trace amounts of [3H]InsP6and an ATP regeneration system (see “Experimental Procedures”). The InsP6 was found to be completely phosphorylated to PP-[3H]InsP5 (Fig.6 A and Ref. 13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). No [PP]2-InsP4 was formed (data not shown and see Ref. 13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Several other inositol polyphosphates, namely inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, have also previously been found not to be significant substrates for this enzyme (13Saiardi A. Erdjument-Bromage H. Snowman A. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). However, Ins(1,3,4,5,6)P5 was not previously tested as a substrate. We now studied this issue, in view of the observation that the yeast kinase phosphorylated both InsP6 and Ins(1,3,4,5,6)P5 (Fig. 4).Figure 6Phosphorylation of [3H] InsP6 and [3H] InsP5 by the mammalian type 1 InsP6 kinase. All incubations were performed as described under “Experimental Procedures.” A depicts an HPLC analysis of reactions containing the type 1 kinase (1 ng/μl) incubated with approximately 5000 dpm of [3H]InsP6 for either 0 min (open circles) or 120 min (closed circles). Bdepicts an HPLC analysis of reactions containing the type 1 kinase (2 ng/μl) incubated for 0 min (open circles) or 120 min (closed circles) with approximately 10,000 dpm of [3H]Ins(1,3,4,5,6)P5. Peak X is a previously unknown product that we have identified as [PP]2-InsP3 (see text). The insetto B shows a time course for the phosphorylation of Ins(1,3,4,5,6)P5 (open circles) to both PP-[3H]InsP4 (closed circles) and peak X (squares) by 2 ng/μl of the kinase. These data are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The type 1 InsP6 kinase phosphorylated Ins(1,3,4,5,6)P5 (Fig. 6 B). TheV max values for Ins(1,3,4,5,6)P5 and InsP6 were very similar (TableII). The affinity of this enzyme for InsP6 was only 5-fold higher than that for Ins(1,3,4,5,6)P5 (Table II). Because levels of both InsP6 and Ins(1,3,4,5,6)P5 in mammalian cells each range from 15 to 50 μm (27Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (153) Google Scholar), we can anticipate that these two substrates will compete for phosphorylation by this enzymein vivo.Table IIKinetic parameters for phosphorylation of InsP5 and InsP6 by the mammalian type 1 and type 2 InsP6 kinasesEnzymeSubstrateK mV maxμmμmol/mg/mintype 1InsP61.2 ± 0.030.31 ± 0.09typ" @default.
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