Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation

Abstract

Schizosaccharomyces pombe extracts synthesize InsP6 (myo-inositol hexaphosphate) from Ins(1,4,5)P3 plus ATP. An S. pombe soluble fraction converts Ins(1,4,5)P3 into Ins(1,4,5,6)P4 and Ins(1,3,4, 5)P4, in a constant ratio of approximately 5:1, and thence to Ins(1, 3,4,5,6)P5 and InsP6. We have purified a soluble Mg2+-dependent kinase of molecular mass approximately 41 kDa that makes Ins(1,4,5, 6)P4 and Ins(1,3,4,5)P4 in the same ratio and also converts Ins(1,4, 5,6)P4 or Ins(1,3,4,5)P4 into Ins(1,3,4,5,6)P5 and InsP6. Of InsP3 isomers other than Ins(1,4,5)P3, only the non-biological molecule Ins(1,4,6)P3 potently 'competed' with all steps in conversion of Ins(1,4,5)P3 into InsP6. Examination of molecular graphics representations allowed us to draw tentative conclusions about the environment needed for an hydroxyl group to be phosphorylated by this kinase and to predict successfully that the purified kinase would phosphorylate the 5-hydroxyl of Ins(1,4,6)P3. S. pombe that have been cultured with [3H]inositol contains a variety of 3H-labelled inositol polyphosphates, with Ins(1,4,5)P3 and InsP6 the most prominent, and the InsP6 concentration quickly increases in hyper-osmotically stressed S. pombe. This yeast therefore contains InsP6 and Ins(1,4,5)P3 as normal constituents, makes more InsP6 when hyper-osmotically stressed and contains a versatile inositol polyphosphate kinase that synthesizes InsP6 from Ins(1,4,5)P3.