Identification of EPI64 as a GTPase-activating protein specific for Rab27A.
Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Because the GTP- or GDP-locked mutant of Rab27A causes perinuclear aggregation of melanosomes, appropriate GTP-GDP cycling of Rab27A is essential for melanosome transport, and certain guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) of Rab27A must be present in melanocytes. However, no such regulators of Rab27A have ever been identified. In this study we developed novel methods of rapidly screening 40 different TBC (Tre2/Bub2/Cdc16) proteins, putative Rab-GAPs, for Rab27A-GAP by: (i) searching for TBC proteins that induce melanosome aggregation in melanocytes; (ii) trapping GTP-Rab27A with a Rab27A effector domain (i.e. the SHD of Slac2-a) in cultured cells that express both Rab27A and TBC proteins; and (iii) measuring in vitro Rab27A-GAP activity. These methods allowed us to identify EPI64, previously characterized as an EBP50-binding protein that contains an orphan TBC domain, as a specific Rab27A-GAP. We further showed that mutations in the catalytic domain of EPI64 caused complete loss of its ability to induce melanosome aggregation. This is the first report of screening for Rab27A-GAP based on functional interactions, and our screening methods can be applied for other uncharacterized TBC proteins.
Itoh
Takashi
T
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Fukuda
Mitsunori
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D020460:Melanosomes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
D002352:Carrier Proteins; C515119:EPI64 protein, mouse; D020690:GTPase-Activating Proteins; C425805:TBC1D10A protein, human; D000075745:rab27 GTP-Binding Proteins; D019204:GTP-Binding Proteins; C066694:RAB27A protein, human; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins