Assay and functional properties of Rab34 interaction with RILP in lysosome morphogenesis

We have recently characterized Rab34 as a new member of the Rab GTPase family based on its ability to regulate lysosomal morphology. Rabbit polyclonal antibody raised against recombinant Rab34 reveals that Rab34 is a 29-kDa protein present both in the cytosol and in the Golgi apparatus. A GTP overlay assay shows that a wild-type and GTP-restricted mutant form of recombinant Rab34 bind GTP in vitro. Yeast two-hybrid interaction screens identify Rab7-interacting lysosomal protein (RILP) as a partner of Rab34. Both GST pull-down experiments and direct binding assays in vitro demonstrate that RILP interacts selectively with the wild-type and GTP-restricted but not GDP-restricted form of Rab34. A key residue (K82) of Rab34 is necessary for interaction with RILP. Expression of EGFP-tagged Rab34 wild-type or GTP-restricted forms in mammalian cells results in redistribution of clustered lysosomes to the peri-Golgi region and this property depends on K82, suggesting that Rab34 regulates lysosome distribution via interaction with RILP. These results suggest that RILP is a common effector shared by Rab7 and Rab34. We describe the methods used in our study.