ARF AND PITP RESTORE GTP-GAMMA-S-STIMULATED PROTEIN SECRETION FROM CYTOSOL-DEPLETED HL-60 CELLS BY PROMOTING PIP2 SYNTHESIS

Background: In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca 2+-regulated secretion. Neutrophils and HL60 cells secrete lysosomal e nzymes from azurophilic granules; this secretion is inhibited by 1% et hanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in prefer ence to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ri bosylation factor) proteins regulate PLD activity and are implicated i n constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells. Results: We h ave used a cell-permeabilization protocol that allows HL60 cells to be come refractory to stimulation with GTP gamma S plus 10 mu M Ca2+ with regard to secretion and PLD activity. Permeabilization with streptoly sin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytoso l, enriched in ARF proteins, restored secretory function and PLD activ ity. The major contaminating protein present in these ARF-enriched fra ctions was identified as phosphatidylinositol transfer protein (PlTP). Unexpectedly, PlTP was also found to restore GTP gamma S-dependent se cretion. Restoration of secretory function was characterized using rec ombinant proteins, rARF1 and rPlTP alpha. and rPlTP beta. The rARF1 pr otein restored both secretory function and PLD activity, whereas PlTP only restored secretory function. However, both ARF and PlTP were capa ble of stimulating phosphatidylinositol bis phosphate (PlP(2)) synthes is. Conclusions: ARF and PlTP restore secretory function in cytosol-de pleted cells when stimulated with GTP gamma S plus Ca2+. We have previ ously shown that PlTP participates in the synthesis of PIP2. In compar ison, 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.