Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from 
the trans-Golgi network to endosomes, then return to the trans-Golgi network for 
another round of enzyme delivery. Wortmannin, an inhibitor of 
phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized 
lysosomal enzymes to lysosomes. We used two independent assays of mannose 
6-phosphate receptor trafficking to determine the precise step that is blocked 
by wortmannin. Using an assay that monitors resialylation of desialylated cell 
surface 300-kDa mannose 6-phosphate receptors, we found that receptor 
endocytosis and transport to the trans-Golgi network were not inhibited by 2 
microM wortmannin. In addition, this concentration of drug had no effect on the 
transport of the mannose 6-phosphate receptor from late endosomes to the 
trans-Golgi network using a system that reconstitutes this transport process in 
cell extracts. Under the same conditions, wortmannin significantly inhibited the 
generation of mature cathepsin D. In addition, the structurally unrelated 
phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when 
added to in vitro endosome-trans-Golgi network transport reactions. These 
experiments demonstrate that the interruption in lysosomal enzyme targeting is 
most likely due to a wortmannin-sensitive process required for the export of 
these receptors from the trans-Golgi network, consistent with the established 
role of phosphatidylinositol 3-kinase in the equivalent transport process in 
Saccharomyces cerevisiae.