EVIDENCE FOR A RECYCLING ROLE FOR RAB7 IN REGULATING A LATE STEP IN ENDOCYTOSIS AND IN RETENTION OF LYSOSOMAL-ENZYMES IN DICTYOSTELIUM-DISCOIDEUM

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has b een implicated in regulating membrane traffic at postinternalization s teps along the endosomal pathway. A cDNA encoding a protein 85% identi cal at the amino acid level to mammalian Rab7 has been cloned from Dic tyostelium discoideum. Subcellular fractionation and immunofluorescenc e microscopy indicated that Rab7 was enriched in lysosomes, postylsoso mes, and maturing phagosomes. Cell lines were generated that overexpre ssed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line i nternalized fluid phase markers and latex beads (phagocytosis) at one- third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell li nes were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic com partments for longer periods of time and were more slowly exocytosed f rom Rab7 T22N cells as compared with control cells. Light microscopy i ndicated that Rab7-expressing cell lines contained morphologically alt ered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles the size of postlysosomes (>2.5 mu m) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, >90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained a n increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normall y, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affect ed in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observ ed for the fluid phase efflux experiments, Rab7 T22N cells oversecrete d alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H+-A TPase from postlysosomes to lysosomes coupled with the efficient relea se of fluid phase from cells.