Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Levels Decrease with Time in Culture

During the stationary phase of growth, after 7 to 12 d in culture, the levels of phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) decreased by 75% in plasma membranes of the red alga Galdieria sulphuraria. Concomitant with the decrease in PtdInsP(2) levels in plasma membranes, there was an increase in PtdInsP(2) in microsomes, suggesting that the levels of plasma membrane PtdInsP(2) are regulated differentially. The decline of PtdInsP(2) in plasma membranes was accompanied by a 70% decrease in the specific activity of PtdInsP kinase and by reduced levels of protein cross-reacting with antisera against a conserved PtdInsP kinase domain. Upon osmotic stimulation, the loss of PtdInsP(2)from the plasma membrane increased from 10% in 7-d-old cells to 60% in 12-d-old cells, although the levels of inositol 1,4,5-trisphosphate (InsP(3)) produced in whole cells were roughly equal at both times. When cells with low plasma membrane PtdInsP(2) levels were osmotically stimulated, a mild osmotic stress (12.5 mM KCl) activated PtdInsP kinase prior to InsP(3) production, whereas in cells with high plasma membrane PtdInsP(2), more severe stress (250 mM KCl) was required to induce an increase in PtdInsP kinase activity. The differential regulation of a plasma membrane signaling pool of PtdInsP(2) is discussed with regard to the implications for understanding the responsive state of cells.