WORTMANNIN-SENSITIVE TRAFFICKING PATHWAYS IN CHINESE-HAMSTER OVARY CELLS - DIFFERENTIAL-EFFECTS ON ENDOCYTOSIS AND LYSOSOMAL SORTING

Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases imp licated in the regulation of cell growth by oncogene products and tyro sine kinase growth factor receptors. The catalytic subunit of the p85/ p110 PI 3'kinase is homologous to VPS-34, a phosphatidylinositol-speci fic lipid kinase involved in the sorting of newly synthesized hydrolas es to the yeast vacuole. This suggests that PI 3'-kinases may play ana logous roles in mammalian cells. We have measured a number of secretor y and endocytic trafficking events in Chinese hamster ovary cells in t he presence of wortmannin, a potent inhibitor of PI S'-kinase. Wortman nin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibiti on of the p85/p110 PI 3'-kinase in intact cells. The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate. Experiments with fluor escent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin. Wortmannin altered the morphology of the re cycling compartment, which was more vesiculated than in untreated cell s. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slow ed by wortmannin, whereas endosomal acidification was unaffected. In c ontrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike p roteins. Wortmannin did induce missorting of the lysosomal enzyme cath epsin D to the secretory pathway, but only at a dose 20-fold greater t han that required to inhibit p85/p110 PI 3'kinase activity or to redis tribute transferrin receptors. Our data demonstrate the presence of wo rtmannin-sensitive enzymes at three distinct steps of the endocytic cy cle in Chinese hamster ovary cells: internalization, transit from earl y endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmann in-sensitive enzymes critical for endocytosis and recycling are distin ct from those involved in sorting newly synthesized lysosomal enzymes.