Wortmannin-sensitive Trafficking Pathways in Chinese Hamster Ovary Cells

Abstract

Phosphatidylinositol (PI) 3′-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors. The catalytic subunit of the p85/p110 PI 3′-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole. This suggests that PI 3′-kinases may play analogous roles in mammalian cells. We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3′-kinase. Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition 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 fluorescent 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 recycling compartment, which was more vesiculated than in untreated cells. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected. In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins. Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3′-kinase activity or to redistribute transferrin receptors. Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes.