ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE IN THE SORTING AND TRANSPORT OF NEWLY SYNTHESIZED LYSOSOMAL-ENZYMES IN MAMMALIAN-CELLS

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product o f the VPS34 gene, in the targeting of newly synthesized proteins to th e vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J, Fry, J. H, Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of V ps34p kinase is significantly reduced by the PI 3-kinase inhibitors wo rtmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack , J. H., and S. D, Emr. 1994. J, Biol. Chem. 269:31552-31562), We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase a ctivity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-max imal inhibition of delivery occurring at 100 nM wortmannin. As a resul t of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Gol gi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery , both wortmannin and LY294002 caused a highly specific dilation of ma nnose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to similar to 1 mu m within 15 min a fter treatment. With increasing time, the inhibitors caused a signific ant yet reversible change in M6PR distribution. By 3 h of treatment, t he swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surfac e. However, M6PRs were still abundant in the TGN. These results are mo st consistent with the interpretation that PI 3-kinase regulates the t rafficking of lysosomal enzymes by interfering with a M6PR-dependent s orting event in the TGN. Moreover, they provide evidence that traffick ing of soluble hydrolases to mammalian lysosomes and yeast vacuoles re ly on similar regulatory mechanisms.