EPI64 interacts with Slp1/JFC1 to coordinate Rab8a and Arf6 membrane trafficking.
Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.
Weill Institute of Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
David E
DE
Hokanson
Anthony P
AP
Bretscher
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D021381:Protein Transport; D014617:Vacuoles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; C431146:SYTL1 protein, human; C425805:TBC1D10A protein, human; C082377:RAB8A protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020691:rab GTP-Binding Proteins