REGULATED fusion of secretory granules with the plasma membrane in sec
retory cells requires ATP, Ca2+ and cytosolic(1-3) as well as membrane
(4) proteins. ATP-dependent steps in Ca2+-activated secretion from PC1
2 cells require three cytosolic PEP proteins (priming in exocytosis pr
oteins, PEP1-3)(5,6), the identity of which will provide insights into
the required ATP-using reactions. PEP3 was recently identified as pho
sphatidylinositol transfer protein (PtdInsTP)(6), and here we report t
hat PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kin
ase (PtdInsP5K), The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequ
ential phosphoinositide recruitment and phosphorylation explains their
synergistic activity in ATP-dependent priming, Moreover, inhibition o
f Ca2+-activated secretion by PtdIns(4,5)P-2-specific antibodies and p
hospholipase C implies that 5-phosphorylated inositides play a novel,
necessary role in the regulated secretory pathway. The results indicat
e that lipid kinase-mediated phosphorylation is an important basis for
ATP use in the exocytotic pathway.