Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role 
for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, 
in the targeting of newly synthesized proteins to the 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 Vps34p kinase is significantly reduced by the PI 
3-kinase inhibitors wortmannin, 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 
activity, wortmannin also inhibits the processing and delivery of newly 
synthesized cathepsin D to lysosomes in mammalian cells with half-maximal 
inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin 
action, newly synthesized, unprocessed cathepsin D is secreted into the media. 
Moreover, after accumulation in the trans-Golgi 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 mannose 6-phosphate receptor (M6PR)-enriched vesicles of the 
prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 
min after treatment. With increasing time, the inhibitors caused a significant 
yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC 
vacuoles were essentially depleted of receptors and, in addition, there was a 
fourfold loss of receptors from the cell surface. However, M6PRs were still 
abundant in the TGN. These results are most consistent with the interpretation 
that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering 
with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence 
that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles 
rely on similar regulatory mechanisms.