title: RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins.
abstract: Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Nottingham
authors.forename: Ian G
authors.initials: IG
authors.lastname: Ganley
authors.forename: Francis A
authors.initials: FA
authors.lastname: Barr
authors.forename: David G
authors.initials: DG
authors.lastname: Lambright
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002457:Cell Extracts; D002460:Cell Line; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; C523295:SGSM2 protein, human; D006160:Guanosine Triphosphate; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins