PHOSPHATIDYLINOSITOL 3-KINASE IS REQUIRED FOR THE FORMATION OF CONSTITUTIVE TRANSPORT VESICLES FROM THE TGN

An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphop rotein (p62) and a 25-kD GTPase, has been shown to be essential for th e cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containi ng exocytic transport vesicle formation from the TGN (Jones, S.M., J.R . Crosby, J. Salamero, and K.E. Howell, 1993. J. Cell Biol. 122:775-78 8). Here the p62(cplx) is identified as a regulatory subunit of a nove l phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associate d PI3-kinase activity is stimulated by activation of the p62(cplx)-ass ociated GTPase, and is specific for phosphatidylinositol (PI) as subst rate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-d erived vesicle formation is indicated by the finding that both lipid k inase activity and the formation of pIgA-R-containing exocytic vesicle s from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 mu M). These findings in dicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essen tial molecules that drive production of this PI(3)P.