Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cbeta 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 diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [gamma-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCbeta (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 degrees C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely 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, and back. This labelled PA was not converted into PI. We conclude that PA derived 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 contact.
Department of Physiology, Rockefeller Building, 1 University St., University College London, London WC1E 6JJ, U.K.
J
J
Whatmore
C
C
Wiedemann
P
P
Somerharju
P
P
Swigart
S
S
Cockcroft
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008055:Lipids; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010712:Phosphatidic Acids / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D054799:Phospholipase C beta; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D008055:Lipids; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D054799:Phospholipase C beta