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Fig. 1. Mapping of the site in Slac2-a responsible for the binding of the globular tail of myosin-Va. (A) Deletion mutants of Slac2-a. Slac2-a consists of four distinct domains: the N-terminal SHD (SHD1 = RBD27, two Cys-based Zn2+-finger motifs, and SHD2; black boxes) (Fukuda et al., 2001a; Fukuda, 2002a; Fukuda, 2002b; Kuroda et al., 2002a), a myosin-Va-GT-binding site (MyoVa-GT; cross-hatched box), a myosin-Va-exon-F-binding site (MyoVa-F; gray box) (Strom et al., 2002; Fukuda and Kuroda, 2002; Nagashima et al., 2002) and an actin-binding site (hatched box) (Fukuda and Kuroda, 2002; Kuroda et al., 2003). The solid lines represent the deletion constructs of T7-tagged Slac2-a. The myosin-Va-GT-binding activity or the myosin-Va-exon-F-binding activity of each mutant is indicated after its name (+ or -) (see Fig. 1B). Bold lines indicate the minimal myosin-Va-GT-binding site of Slac2-a (amino acid residues 147-240), which is completely different from the myosin-Va-exon-F-binding site (shaded box) (Strom et al., 2002; Fukuda and Kuroda, 2002; Nagashima et al., 2002). The sequences at the bottom represent the myosin-Va-GT-binding site of human and mouse Slac2-a. Residues in the sequences that are conserved and similar are shown against a black background and a shaded background, respectively. The amino acid numbers are indicated at the right-hand side of each sequence. (B) Mapping of the site in Slac2-a responsible for the binding of the GT of 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 FLAG-myosin-Va-GT, and proteins trapped with the beads were analyzed by immunoblotting with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution) (top panel; 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 mutant proteins had been loaded (bottom panel; Blot, anti-T7; IP, anti-T7). Note that myosin-Va-GT bound amino acids 147-240 of Slac2-a, adjacent to the SHD. Although the apparent molecular mass of the T7-Slac2-a mutants almost corresponded to their calculated molecular weight, several additional bands with higher molecular mass were observed in Slac2-a-
146/321 mutant (asterisks in lane 4). These bands were probably produced by certain post-translational modifications caused by the truncation of Slac2-a protein. One of the possible modifications is fatty-acylation, which often contributes to the formation of a SDS-insensitive oligomer on SDS-polyacrylamide gel (Fukuda et al., 2001b). In addition, Slac2-a-400 and -240 mutants contained some degradation products (lanes 5 and 6). The positions of the molecular mass markers (·10-3) are shown on the right. (C) Rab27A enhanced myosin-Va-GT binding to Slac2-a. The purified T7-Slac2-a coupled with the beads was incubated with recombinant FLAG-myosin-Va-GT in the presence and absence of HA-Rab27A. The positions of the molecular mass markers (·10-3) are shown on the left.
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