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Screening for target Rabs of TBC (Tre‐2/Bub2/Cdc16) domain‐containing proteins based on their Rab‐binding activity
Author(s) -
Itoh Takashi,
Satoh Megumi,
Kanno Eiko,
Fukuda Mitsunori
Publication year - 2006
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2006.00997.x
Subject(s) - rab , biology , gtpase , gtpase activating protein , yeast , microbiology and biotechnology , biochemistry , g protein , signal transduction
It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase‐activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab‐GAP activity. In this study we established panels of the GTP‐fixed form of 60 different Rabs constructed in pGAD‐C1, a yeast two‐hybrid bait vector. We also constructed a yeast two‐hybrid prey vector (pGBDU‐C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP‐fixed Rabs and 40 TBC proteins by yeast two‐hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5‐like with Rab10). Measurement of in vitro Rab‐GAP activity revealed that OATL1 and Evi5‐like actually possess significant Rab2A‐ and Rab10‐GAP activity, respectively, but that FLJ12085 do not display Rab5A‐GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab‐GAP domains, but that there is little correlation between the Rab‐binding activity and Rab‐GAP activity of other TBC proteins.