RRC ID 48776
Author Itoh T, Fukuda M.
Title Identification of EPI64 as a GTPase-activating protein specific for Rab27A.
Journal J Biol Chem
Abstract 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.
Volume 281(42)
Pages 31823-31
Published 2006-10-20
DOI 10.1074/jbc.M603808200
PII M603808200
PMID 16923811
MeSH Amino Acid Sequence Animals COS Cells Carrier Proteins / chemistry Carrier Proteins / physiology* Catalytic Domain Chlorocebus aethiops GTP-Binding Proteins / chemistry* GTPase-Activating Proteins / chemistry GTPase-Activating Proteins / physiology* Humans Melanosomes / metabolism Mice Molecular Sequence Data Sequence Homology, Amino Acid rab GTP-Binding Proteins / chemistry* rab GTP-Binding Proteins / metabolism rab27 GTP-Binding Proteins
IF 4.106
Times Cited 67
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pEGFP-C1-human TBC1D1 (RDB14982) pEGFP-C1-human TBC1D2A (RDB14983) pEGFP-C1-human TBC1D3 (RDB14985) pEGFP-C1-human TBC1D4 (RDB14986) pEGFP-C1-human TBC1D5 (RDB14987) pEGFP-C1-human TBC1D6 (RDB14988) pEGFP-C1-human TBC1D8A (RDB14990) pEGFP-C1-human TBC1D8B (RDB14991) pEGFP-C1-human TBC1D9A (RDB14992) pEGFP-C1-human TBC1D9B (RDB14993) pEGFP-C1-human TBC1D10A (RDB14994) pEGFP-C1-human TBC1D10B (RDB14995) pEGFP-C1-human TBC1D10C (RDB14996) pEGFP-C1-human TBC1D11 (RDB14997) pEGFP-C1-human TBC1D12 (RDB14998) pEGFP-C1-human TBC1D13 (RDB15000) pEGFP-C1-human TBC1D14 (RDB15001) pEGFP-C1-human TBC1D15 (RDB15002) pEGFP-C1-human TBC1D16 (RDB15003) pEGFP-C1-human TBC1D17 (RDB15004) pEGFP-C1-human TBC1D19 (RDB15006) pEGFP-C1-human TBC1D20 (RDB15007) pEGFP-C1-human TBC1D21 (RDB15008) pEGFP-C1-human TBC1D22A (RDB15009) pEGFP-C1-human TBC1D23 (RDB15011) pEGFP-C1-human TBC1D24 (RDB15012) pEGFP-C1-human TBC1D25 (RDB15013) pEGFP-C1-human TBC1D26 (RDB15014) pEGFP-C1-human TBC1D30 (RDB15017) pEGFP-C1-human RUTBC1 (RDB15018) pEGFP-C1-human RUTBC2 (RDB15019) pEGFP-C1-human RUTBC3 (RDB15020) pEGFP-C1-human EVI5 (RDB15021) pEGFP-C1-human EVI5L (RDB15022) pEGFP-C1-human USP6 (RDB15024) pEGFP-C1-human USP6NL (RDB15025) pEGFP-C1-human RAB3GAP1 (RDB15026).