RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36

Ryan M Nottingham,Ganesh V Pusapati,Ian G Ganley,Francis A Barr,David G Lambright,Suzanne R Pfeffer

doi:10.1074/jbc.m112.362558

Abstract

Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.

Highlights

Rab GTPases control membrane traffic, and identification of Rab regulators is incomplete
As a starting point to find regulators of Rab9A, we used a yeast two-hybrid screen consisting of all TBC domain-containing proteins in the human genome as prey against a comprehensive library of hydrolysis-deficient Rab GTPases as bait [14, 25]
We have shown that a predicted RabGAP protein, RUTBC2, is a novel Rab9A effector

Summary

Background

Rab GTPases control membrane traffic, and identification of Rab regulators is incomplete. Results: RUTBC2 binds Rab9A, enhances GTP hydrolysis by Rab and Rab in vitro, and is a GAP for Rab in cells. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A both in vitro and in cultured cells but is not a GAP for Rab9A. Membrane-associated RUTBC2 co-localizes with Rab, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab protein These data show that RUTBC2 can act as a Rab GAP in cells and suggest that RUTBC2 links Rab9A function to Rab function in the endosomal system. Rab GTPases are master regulators of membrane trafficking [1] They catalyze the formation of functional membrane microdomains by recruiting effectors to specific membranes while in their GTP-bound states. Rab is a bona fide substrate for RUTBC2

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION