GAP domain alignment and Y2H analysis. (A) Sequence alignment of the GAP domains of Leep2A, Leep2B, human Rap1GAP, human RalGAPA1, and human RalGAPB. Red box indicates the catalytic motif. The catalytic asparagine residues conserved in Leep2A, human Rap1GAP, and human RalGAPA1 are highlighted in red. (B) Yeast two-hybrid screen of the active forms of Ras, Rac, and Rab subfamily GTPases for interaction with the GAP domain of Leep2A. The GAP domain was fused to the Gal4 activation domain (AD) and small GTPases to the Gal4 binding domain (BD). Yeast was transformed with the indicated constructs and selected for the presence of prey and bait plasmids by growth on double-dropout agar plate lacking leucine and tryptophan (-LW). Interactions were assayed by growth on quadruple-dropout agar plate additionally lacking histidine and adenine (-LWHA). The GAP-RacM interaction was non-specific since yeast expressing RacM alone also grew on the quadruple-dropout plate.