WORTMANNIN CAUSES MISTARGETING OF PROCATHEPSIN-D - THE INVOLVEMENT OFA PHOSPHATIDYLINOSITOL 3-KINASE IN VESICULAR TRANSPORT TO LYSOSOMES

At present little is known of the biochemical machinery controlling tr ansport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomy ces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested t he possibility that a homologous enzyme might be involved in the equiv alent step in mammalian cells. Using the PI3-K inhibitors wortmannin a nd LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, wi th half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affec ted, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wort mannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibito rs of PI3-K. I conclude that these data implicate such an enzyme in th e trafficking of M6P-receptor-ligand complexes from the TGN towards ly sosomes.