PHOSPHATIDYLINOSITOL 3-KINASE IS NOT REQUIRED FOR RECYCLING OF MANNOSE 6-PHOSPHATE RECEPTORS FROM LATE ENDOSOMES TO THE TRANS-GOLGI-NETWORK

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrol ases from the trans-Golgi network to endosomes, then return to the tra ns-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delive ry of newly synthesized lysosomal enzymes to lysosomes. We used two in dependent assays of mannose 6-phosphate receptor trafficking to determ ine the precise step that is blocked by wortmannin. Using an assay tha t monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transpor t to the trans-Golgi network were not inhibited by 2 mu M 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 stru cturally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi ne twork transport reactions. These experiments demonstrate that the inte rruption in lysosomal enzyme targeting is most Likely due to a wortman nin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosp hatidylinositol 3-kinase in the equivalent transport process in Saccha romyces cerevisiae.