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First published online January 16, 2004
doi: 10.1242/10.1242/jcs.00891

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Missense mutations in the globular tail of myosin-Va in dilute mice partially impair binding of Slac2-a/melanophilin

Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

View larger version (27K): [in a new window]   Fig. 2. Differential effects of ionic strength on the Slac2-a·myosin-Va complex according to whether exon F is present. COS-7 cell lysates containing FLAG-myosin-Va-GT (or FLAG-myosin-Va-F-GT) (A) or containing FLAG-brain myosin-Va-tail (or FLAG-MC myosin-Va-tail) (B) were incubated with T7-Slac2-a beads in the presence of the concentrations of NaCl indicated. Proteins that bound to the beads were first analyzed with HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG, top panels), and then with HRP-conjugated anti-T7 tag antibody (Blot: anti-T7, bottom panels). Note that the binding of the MC-type myosin-Va to Slac2-a was resistant to ionic strength (up to 750 mM NaCl) (lanes 5-8), whereas binding of brain myosin-Va to Slac2-a was highly sensitive to ionic strength (lanes 1-4). (C) Tail constructs of brain-type and MC-type myosin-Va used in this study.

View larger version (35K): [in a new window]   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.

View larger version (64K): [in a new window]   Fig. 6. Deletion of the myosin-Va-GT-binding site of Slac2-a creates a dilute-like phenotype in wild-type melanocytes. Melan-a cells were transfected with a vector encoding GFP-tagged wild-type Slac2-a (AE) or mutant GFP-Slac2-a-GT (F-J). After the cells were fixed and permeabilized, they were stained with anti-Rab27A (green) and anti-myosin-Va (red) antibodies followed by Alexa Fluor secondary antibody conjugates as described previously (Kuroda et al., 2002a; Kuroda et al., 2003). The cells were examined for fluorescence of GFP-Slac2-a proteins (A and F), Rab27A (B and G) and myosin-Va (C and H) by confocal microscopy. Bright-field images (E and J) show the melanosome distribution in the cells, and the mutant GFPSlac2-a-GT-expressing cell is outlined in yellow (J). D and I are merged images of Rab27A and myosin-Va. Note that expression of GFP-Slac2-a-GT induced melanosome aggregation in the perinuclear region (J), segregation of myosin-Va from Rab27A (I) and attenuation of myosin-Va-immunoreactivity (H), whereas the wild-type Slac2-a-expressing cell exhibited peripheral melanosome distribution (E), with both Rab27A and myosin-Va being colocalized on melanosomes (D). Bars, 10 µm.

View larger version (33K): [in a new window]   Fig. 3. Slac2-a bound MC myosin-Va with higher affinity than it bound brain myosin-Va. (A) Recombinant MC myosin-Va-tail (left panel) and brain myosin-Va-tail (right panel) were systematically diluted as indicated, and diluted samples were incubated with beads coupled with T7-Slac2-a protein (0.25 µg). Proteins bound to the beads were analyzed by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (top panels). The bottom panels indicate the input FLAG-myosin-Va-tail (1/50 volume of the reaction mixture) visualized with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution). The positions of the FLAG-myosin-Va-tail and T7-Slac2-a proteins are indicated by the closed arrowhead and open arrowhead, respectively. (B) Bands of myosin-Va-tail on gels were captured and quantified as described in Materials and Methods. The EC50 value of 0.15 for the Slac2-a·MC myosin-Va-tail interaction was calculated with GraphPad PRISM software (broken line). Bars indicate the standard error of three independent experiments. Open circles, Slac2-a·MC myosin-Va-tail interaction; closed circles, Slac2-a·brain myosin-Va-tail interaction; open squares, Slac2-a-GT·MC myosin-Va-tail interaction; and closed squares, Slac2-a-GT·brain myosin-Va-tail interaction. Judging from the intensity of the bands in A, one Slac2-a molecule binds approximately two molecules of myosin-Va-tail. The results shown are representative of three independent experiments.

View larger version (63K): [in a new window]   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.

View larger version (32K): [in a new window]   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).