Resynthesis of phosphatidylinositol in permeabilized neutrophils followingphospholipase C beta activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylgl ycerol (DG) is converted into phosphatidate (PA). Two enzymes located at th e endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be fol lowed in permeabilized cells. In the presence of [gamma-P-32]ATP, DG genera ted by PLC activation accumulates label when converted into PA. The P-32-la belled PA is subsequently converted into labelled PI. The formation of labe lled PI reports the arrival of labelled PA from the plasma membrane to the ER, Cytosol-depleted, permeabilized human neutrophils are capable of PI res ynthesis following stimulation of PLC beta (in the presence of phosphatidyl inositol-transfer protein), provided that CTP and inositol are also present . We also found that wortmannin, an inhibitor of endocytosis, or cooling th e cells to 15 degrees C did not stop PI resynthesis. We conclude that PI re synthesis is dependent neither on vesicular transport mechanisms nor on fre ely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, a nd back. This labelled PA was not converted into PI. We conclude that PA de rived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane co ntact.