MUTANT RAB7 CAUSES THE ACCUMULATION OF CATHEPSIN-D AND CATION-INDEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR IN AN EARLY ENDOCYTIC COMPARTMENT

Stable BHK cell lines inducibly expressing wild-type or dominant negat ive mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rabi7-regulated pathway in lysosome biogenesis. Expressi on of mutant rab7N125I protein induced a dramatic redistribution of ca tion-independent mannose 6-phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes, Under these c ircumstances similar to 50% of the total receptor and several lysosoma l hydrolases cofractionated with light membranes containing early endo some and Golgi markers. Late endosomes and lysosomes were contained ex clusively in well-separated, denser gradient fractions. Newly synthesi zed CI-MPR and cathepsin D were shown to traverse through an early end ocytic compartment, and functional rab7 was crucial for delivery to la ter compartments, This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions contain ing light membranes. In addition, both were sensitive to HRP-DAB-media ted cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired, Using similar criteria, the ly sosomal membrane glycoprotein 120 was not found accumulated in an earl y endocytic compartment. The data are indicative of a post-Golgi diver gence in the routes followed by different lysosome-directed molecules.