The inositol hexakisphosphate kinase family. Catalytic flexibility and function in yeast vacuole biogenesis.
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 (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta 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 InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), 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 InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.
Saiardi
A
A
Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Caffrey
J J
JJ
Snyder
S H
SH
Shears
S B
SB
D000818:Animals; D002384:Catalysis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008322:Mammals; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014617:Vacuoles / Q000235:genetics / Q000502:physiology*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C115134:IP6K1 protein, human; C098863:KCS1 protein, S cerevisiae; C099438:inositol hexakisphosphate kinase