Assay and Functional Properties of Rab34 Interaction with RILP in Lysosome Morphogenesis

Abstract 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.