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 (InsP₆) to diphosphoinositol pentakisphosphate, a 'high energy' candidate regulator of cellular trafficking. We have now studied the significance of InsP₆ kinase activity in Saccharomyces 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 InsP₆ kinases were identified; inositol pentakisphosphate (InsP₅) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP₄), which was further metabolized to a novel compound, tentatively identified as bisdiphosphoinositol 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) = 1.2μM) 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.