EVIDENCE FOR SUBSTRATE-CYCLING OF 3-PHOSPHORYLATED, 3,4-PHOSPHORYLATED, 4-PHOSPHORYLATED, AND 4,5-PHOSPHORYLATED PHOSPHATIDYLINOSITOLS IN PLANTS

Short-term P-32 labelling and enzymic dissection of inositol phospholipids was used to study the turnover of 3-, 3,4-, 4-, and 4,5-phosphorylated phosphatidylinositols in the plant Spirodela polyrhiza L. Analysis of label in the whole headgroup reveals that phosphatidylinositol 3- and 4-monophosphates (PtdIns3P and PtdIns4P) and phosphatidylinositol 3,4- and 4,5-bisphosphates [PtdIns(3,4)P-2 and PtdIns(4,5)P-2] all turn over with a half-life of approximately 2-5 h. Analysis of the labelling of individual phosphomonoesters and phosphodiesters of these lipids indicates a rapid equilibration of label between the 4- and 5-monoester phosphates of PtdIns(4,5)P-2 within 5 h and largely independent of changer; of labelling in the diester. We observed substantially slower equilibration of label (within similar to 27 h) between the monoester and diester of PtdIns4P. These studies therefore indicate that PtdIns4P and PtdIns(4,5)P-2 participate in substrate-cycling reactions, evidence for which has been described experimentally only in erythrocytes, and give confirmation in vivo of the previous detection of inositol phospholipid phosphomonoesterase activity. Similar analyses of label in PtdIns3P and PtdIns(3,4)P-2 reveal the likely participation of these molecules in substrate cycles and hence for the first time the presence of PtdIns3P 3-phosphatase and PtdIns(3,4)P-2 4-phosphatase activities in plants. PtdIns3P and PtdIns(3,4)P-2 undergo turnover at rates similar to those of PtdIns4P and PtdIns(4,5)P-2 Estimates are made of the relative sizes of the pools of phospholipid participating in the turnover process.