Abstract
There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.
Highlights
Phospholipase D (PLD)-derived phosphatidic acid (PA) is suggested to function in exocytosis
A detailed analysis of fusion parameters using a range of reagents to both block PA and inhibit PLD functions confirmed low level PLD activity associated with CV and a substantially higher activity associated with the PM
A low basal PLD activity and limited PA generation in the CV membrane indicates that PA is highly localized at specific sites of the membrane that are critical for CV docking and fusion [18, 19, 33]
Summary
Phospholipase D (PLD)-derived phosphatidic acid (PA) is suggested to function in exocytosis. Results: PLD activity on the plasma membrane was higher than on vesicles. There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca2؉-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The effects of phospholipase D activity and its product phosphatidic acid on the Ca2؉ sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se
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