Fig. 5. Deletion of the myosin-Va-GT-binding site of Slac2-a impairs binding to brain myosin-Va. (A) A deletion mutant of Slac2-a lacking the myosin-Va-GT-binding site (MyoVa-GT; cross-hatched box) (GT). Rab27A-binding, MC myosin-Va-tail-binding, and brain myosin-Va-tail-binding activity is indicated after their names (+ or -) (see Fig. 5B). The amino acid numbers are indicated at both sides of each line. (B) Effect of deletion of the myosin-Va-GT-binding site of Slac2-a on formation of a tripartite protein complex (Rab27A·Slac2-a·myosin-Va). Purified T7-Slac2-a mutants coupled with anti-T7 tag antibody-conjugated agarose (Fukuda and Kuroda, 2002) were incubated with COS-7 cell lysates containing HARab27A and FLAG-brain myosin-Va-tail or FLAG-MC myosin-Va-tail, and proteins trapped with the beads were analyzed by immunoblotting with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution) (third panel; Blot: anti-FLAG, IP: anti-T7) and HRP-conjugated anti-HA tag antibody (1/10,000 dilution) (fourth panel; Blot: anti-HA, IP: anti-T7). The same blots were then stripped and reprobed with HRP-conjugated anti-T7 tag antibody (1/10,000 dilution) to ensure that the same amounts of T7-Slac2-a mutant proteins had been loaded (bottom panel; Blot: anti-T7, IP: anti-T7). Input means 1/80 volume of the reaction mixture (top and second panels). Note that the Slac2-a-GT mutant specifically impaired brain myosin-Va-tail binding activity (lanes 1 and 2), but that it normally interacted with Rab27A and MC myosin-Va-tail (lanes 4 and 5).