Measurement of Rab35 Activity with the GTP-Rab35 Trapper RBD35
Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.
Key wordsRab35 RBD35 RUSC2 RUN domain Neurite outgrowth
We thank Megumi Aizawa for technical assistance and members of the Fukuda Laboratory for valuable discussions. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology (MEXT) of Japan (to M.F. and N.O.) and by a grant from the Daiichi-Sankyo Foundation of Life Science (to M.F.). H.K. was supported by the Japan Society for the Promotion of Science (JSPS) and by the International Advanced Research and Education Organization of Tohoku University (IAREO).