INOSITOL HEXAKISPHOSPHATE IN SCHIZOSACCHAROMYCES-POMBE - SYNTHESIS FROM INS(1,4,5)P-3 AND OSMOTIC REGULATION

Schizosaccharomyces pombe extracts synthesize InsP(6) (myo-inositol he xaphosphate) from Ins(1,4,5)P-3 plus ATP. An S. pombe soluble fraction converts Ins(1,4,5)P-3 into Ins(1,4,5,6)P-4 and Ins(1,3,4,5)P-4, in a constant ratio of approximate to 5:1, and thence to Ins(1,3,4,5,6)P-5 and InsP(6). We have purified a soluble Mg2+-dependent kinase of mole cular mass approximate to 41 kDa that makes Ins(1,4,5,6)P-4 and Ins(1, 3,4,5)P-4 in the same ratio and also converts Ins(1,4,5,6)P, or Ins(1, 3,4,5)P, into Ins(1,3,4,5,6)P, and InsP(6). Of InsP(3) isomers other t han Ins(1,4,5)P-3, only the nonbiological molecule Ins(1,4,6)P-3 poten tly 'competed' with all steps in conversion of Ins(1,4,5)P-3 into InsP (6). Examination of molecular graphics representations allowed us to d raw 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)P-3. S. pombe that have been cultured with [H-3]inositol contains a variety of H-3-labelled inositol polyphosphates, with Ins(1,4,5)P-3 and InsP(6) the most prominent, and the InsP(6) concentration quickly increases in hyper-osmotically stressed S. pombe. This yeast therefore contains InsP(6) and Ins(1,4,5)P-3 as normal constituents, makes more InsP(6) when hyper-osmotically stressed and contains a versatile inos itol polyphosphate kinase that synthesizes InsP(6) from Ins(1,4,5)P-3.