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 phospholi pids was used to study the turnover of 3-, 3,4-, 4-, and 4,5-phosphory lated phosphatidylinositols in the plant Spirodela polyrhiza L. Analys is of label in the whole headgroup reveals that phosphatidylinositol 3 - and 4-monophosphates (PtdIns3P and PtdIns4P) and phosphatidylinosito l 3,4- and 4,5-bisphosphates [PtdIns(3,4)P-2 and PtdIns(4,5)P-2] all t urn over with a half-life of approximately 2-5 h. Analysis of the labe lling of individual phosphomonoesters and phosphodiesters of these lip ids indicates a rapid equilibration of label between the 4- and 5-mono ester phosphates of PtdIns(4,5)P-2 within 5 h and largely independent of changer; of labelling in the diester. We observed substantially slo wer equilibration of label (within similar to 27 h) between the monoes ter and diester of PtdIns4P. These studies therefore indicate that Ptd Ins4P and PtdIns(4,5)P-2 participate in substrate-cycling reactions, e vidence for which has been described experimentally only in erythrocyt es, and give confirmation in vivo of the previous detection of inosito l phospholipid phosphomonoesterase activity. Similar analyses of label in PtdIns3P and PtdIns(3,4)P-2 reveal the likely participation of the se molecules in substrate cycles and hence for the first time the pres ence of PtdIns3P 3-phosphatase and PtdIns(3,4)P-2 4-phosphatase activi ties 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 t urnover process.