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ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis.
730 - 738.
Background: In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca2+-regulated secretion. Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells.Results: We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 mu M Ca2+ with regard to secretion and PLD activity. Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity. The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PlTP). Unexpectedly, PlTP was also found to restore GTP gamma S-dependent secretion. Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPlTP alpha. and rPlTP beta. The rARF1 protein restored both secretory function and PLD activity, whereas PlTP only restored secretory function. However, both ARF and PlTP were capable of stimulating phosphatidylinositol bis phosphate (PlP(2)) synthesis.Conclusions: ARF and PlTP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+. We have previously shown that PlTP participates in the synthesis of PIP2. In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5-kinase, the enzyme responsible for PlP(2) synthesis. We propose that ARF and PlTP both restore exocytosis by a common mechanism - promoting PlP(2) synthesis.
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