Rab4 is an essential regulator of lysosomal trafficking in trypanosomes

Rapid endocytosis and recycling of surface proteins are important processes common to most nucleated eukaryotic cells. The best characterized membrane recycling routes are mediated by the small GTPases Rab4 and Rab11, but the precise roles that these pathways play have not been fully elucidated. The protozoan Trypanosoma brucei has a highly developed endocytic system that is similar to that found in metazoans, albeit with an accelerated rate of membrane turnover. We have used this organism to investigate the function of the trypanosome orthologue of Rab4 (TbRAB4) by a combination of RNA interference, microscopy, and quantitative trafficking assays. RNA interference-mediated suppression of TbRAB4 expression inhibited the growth of trypanosomes without affecting receptor-mediated endocytosis or ligand recycling. Ultrastructural analysis indicated a major defect in membrane transport events. The accumulation of fluorescent dextran, a fluid-phase marker, was blocked in cells lacking TbRAB4 protein. Since most fluid-phase markers are transported to the lysosome in T. brucei, the effects of TbRAB4 RNA interference on lysosomal function were investigated. By immunofluorescence, the major lysosomal protein p67 became progressively dispersed in cells lacking the TbRAB4 protein. Pulse-chase analysis demonstrated that initial proteolytic cleavage and glycan processing of p67 were unaffected but that cells failed to accumulate the later p67 proteolyzed products associated with the lysosome. To confirm the role of TbRAB4 in lysosomal trafficking, a constitutively active mutant, TbRAB4(QL), was expressed. TbRAB4(QL) was closely associated with an enlarged multivesicular body that contained p67. In addition, cells expressing TbRAB4(QL) showed increased fluid-phase uptake when compared with the parental line. Taken together, these data suggest that TbRAB4 is involved in regulation of fluid-phase traffic to the lysosome in T. brucei but not in receptor-mediated endocytosis or recycling. These data have implications for the role of Rab4 in other cell systems.