Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs

Abstract

The Rab family belongs to the Ras-like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S-transferase (GST) pull-down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab-binding proteins we identified, mKIAA1055/TBC1D2B (Rab22-binding protein), GAPCenA/TBC1D11 (Rab36-binding protein) and centaurin beta2/ACAP2 (Rab35-binding protein), are GTPase-activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab-GAP (Tre-2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin beta2 binds GTP-Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin beta2 did not exhibit any Rab35-GAP activity in vitro, the Rab35-binding ANKR domain of centaurin beta2 was found to be required for its plasma membrane localization and regulation of Rab35-dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.