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.
Tuanlao
T
Wang
Wanjin
W
Hong
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; C423317:RILP protein, human; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; C474116:RAB39A protein, human; D020691:rab GTP-Binding Proteins