Phosphatidylinositol 3–Kinase Is Required for the Formation of Constitutive Transport Vesicles from the TGN

Abstract

An 85-kD cytosolic complex (p62cplx), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775–788). Here the p62cplx is identified as a regulatory subunit of a novel phosphatidylinositol 3–kinase (PI3-kinase). This p62cplx-associated PI3-kinase activity is stimulated by activation of the p62cplx-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62cplx-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R–containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 μM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62cplx-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.