Fig. 4. Effect of dilute missense mutations in the globular tail of myosin-Va on Slac2-a binding. (A) Sequence alignment of the GT of the mouse myosin-V family (myosin-Va, Vb and Vc). Residues in the sequences that are conserved and similar are shown against a black background and a shaded background, respectively. The Af6 homology domain at the C-terminal domain of the globular tail is indicated by the box. The asterisks indicate the point mutations observed in dilute mice (Huang et al., 1998). The # sign indicates the Ca²⁺ calmodulin-dependent protein kinase II phosphorylation site of myosin-Va (Karcher et al., 2001). (B) Interaction of Slac2-a with the first part of the globular tail (lane 1; Af6), but not the Af6 homology domain (lane 2), of myosin-Va. (C) Effect of dilute missense mutations on the interaction between T7-Slac2-a and FLAG-myosin-Va-GT. Note that the I1510N, M1513K and D1519G mutations almost completely abrogated Slac2-a binding activity (lanes 2-4), whereas the S1650E mutation, which mimics a phosphorylated form, had no effect at all (lane 6). (D) Effect of dilute missense mutations on the interaction between T7-Slac2-a and FLAG-myosin-Va-F-GT. Note that in the presence of an MC-specific exon (exon F) all the one-point mutants bound Slac2-a, the same as the wild-type protein did. Purified T7-Slac2-a coupled with anti-T7 tag antibody-conjugated agarose (Fukuda and Kuroda, 2002) was incubated with COS-7 cell lysates containing FLAG-myosin-Va mutants, and the proteins trapped with the beads were analyzed by immunoblotting with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution) (middle panels; Blot, anti-FLAG; 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 proteins had been loaded (bottom panels; Blot, anti-T7; IP, anti-T7). Input means 1/80 volume of the reaction mixture (top panels). The positions of the molecular mass markers (·10^-3) are shown on the left.