Rab9 regulation of the Rab GTPase activating protein, RUTBC1

Abstract

Rab GTPases are master regulators of membrane trafficking. Rab GTPase activating proteins (GAPs) enhance a Rab's slow, intrinsic rate of GTP hydrolysis, thereby inactivating them. A so‐called TBC domain is the catalytic domain found in most Rab GAPs. The goal of this study is to determine the functions of RUTBC1 and RUTBC2, which were identified by two hybrid screen as TBC domain‐containing proteins that bind Rab9. Rab9 is required for recycling of mannose 6‐phosphate receptors (MPRs) from endosomes to the Golgi complex. Over‐expression of RUTBC1 and RUTBC2 destabilized MPRs and increased lysosomal enzyme secretion, consistent with Rab9 regulation. Biochemical screening of 33 Rab GTPases revealed that RUTBC1 is not a GAP for Rab9 but rather acts on the Golgi‐localized, Rab33B with a kcat/Km value of ~2800 M−1 s−1; RUTBC2 is most active on Rab17. Moreover, Rab9 binds RUTBC1 and RUTBC2 at a site far from their catalytic domains and does not competitively inhibit RUTBC1 GAP activity on Rab33B. These results suggest a novel mode of regulation where Rab9 in some way regulates a GAP for another Rab protein. This may help maintain the identity of a Rab‐specific membrane microdomain to inactivate other Rabs should they migrate into a Rab9‐enriched compartment. Further work will explore the significance of this cross‐talk between Rab proteins that mediate distinct membrane traffic events. Research was supported by NIH R37 DK37332.