Abstract
The well-known coat-color mutant mouse dilute exhibits a defect in melanosome transport, and although various mutations in the myosin-Va gene, which encodes an actin-based motor protein, have been identified in dilute mice, why missense mutations in the globular tail of myosin-Va, a putative cargo-binding site, cause the dilute phenotype (i.e. lighter coat color) has never been elucidated. In this study we discovered that missense mutations (I1510N, M1513K and D1519G) in the globular tail (GT) of myosin-Va partially impair the binding of Slac2-a/melanophilin, a linker protein between myosin-Va and Rab27A on the melanosome. The myosin-Va-GT-binding site in Slac2-a was mapped to the region (amino acids 147-240) adjacent to the N-terminal Rab27A-binding site, but it is distinct from the myosin-Va-exon-F-binding site (amino acids 320-406). The myosin-Va-GT.Slac2-a interaction was much weaker than the myosin-Va-exon-F.Slac2-a interaction. The missense mutations in the GT found in dilute mice abrogated only the myosin-Va-GT.Slac2-a interaction and had no effect on the myosin-Va-exon-F.Slac2-a interaction. We further showed that expression of green fluorescence protein-tagged Slac2-a lacking the myosin-Va-GT-binding site (DeltaGT), but not the wild-type Slac2-a, severely inhibits melanosome transport in melan-a cells, especially at the melanosome transfer step from microtubles to actin filaments (i.e. perinuclear aggregation of melanosomes). On the basis of our findings, we propose that myosin-Va interacts with Slac2-a.Rab27A complex on the melanosome via two distinct domains, both of which are essential for melanosome transport in melanocytes.
Highlights
IntroductionGenetic analysis of coat-color mutant mice (dilute, ashen and leaden) (Moore et al, 1988; Mercer et al, 1991; Wilson et al, 2000; Matesic et al, 2001; Hume et al, 2002; Provance et al, 2002) and patients with Griscelli syndrome (Pastural et al, 1997; Menasché et al, 2000; Bahadoran et al, 2001; Menasché et al, 2003; Bahadoran et al, 2003), as well as recent biochemical studies of their gene products (myosin-Va, Rab27A, and Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2 domains-a)/melanophilin, respectively) (Fukuda et al, 2002; Wu et al, 2002a; Strom et al, 2002; Menasché et al, 2003; Bahadoran et al, 2003), have provided evidence that a tripartite protein complex formed by myosinVa, Slac2-a and Rab27A is essential for melanosome transport from the perinuclear region of melanocytes to their actin-rich cell periphery (for a review, see Marks and Seabra, 2001; Hammer and Wu, 2002; Fukuda, 2002a)
The missense mutations in the globular tail (GT) found in dilute mice abrogated only the myosin-Va-GT·Slac2-a interaction and had no effect on the myosin-Va-exon-F·Slac2-a interaction
We further showed that expression of green fluorescence protein-tagged Slac2-a lacking the myosin-Va-GT-binding site (∆GT), but not the wild-type Slac2-a, severely inhibits melanosome transport in melan-a cells, especially at the melanosome transfer step from microtubles to actin filaments
Summary
Genetic analysis of coat-color mutant mice (dilute, ashen and leaden) (Moore et al, 1988; Mercer et al, 1991; Wilson et al, 2000; Matesic et al, 2001; Hume et al, 2002; Provance et al, 2002) and patients with Griscelli syndrome (Pastural et al, 1997; Menasché et al, 2000; Bahadoran et al, 2001; Menasché et al, 2003; Bahadoran et al, 2003), as well as recent biochemical studies of their gene products (myosin-Va, Rab27A, and Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2 domains-a)/melanophilin, respectively) (Fukuda et al, 2002; Wu et al, 2002a; Strom et al, 2002; Menasché et al, 2003; Bahadoran et al, 2003), have provided evidence that a tripartite protein complex formed by myosinVa, Slac2-a and Rab27A is essential for melanosome transport from the perinuclear region of melanocytes to their actin-rich cell periphery (for a review, see Marks and Seabra, 2001; Hammer and Wu, 2002; Fukuda, 2002a). The interaction between Slac2-a and myosin-Va has been shown to be regulated by a melanocyte (MC)-specific alternative splicing in the tail domain of myosin-Va (Seperack et al, 1995; Huang et al, 1998; Wu et al, 2002a; Wu et al, 2002b): Slac2-a strongly interacts with the MC-type myosinVa containing the MC-specific exon F (– exon B, + exon D and + exon F), and weakly with a brain-type myosin-Va lacking exon F (+ exon B, – exon D, and – exon F) (Fukuda et al, 2002; Wu et al, 2002a; Fukuda and Kuroda, 2002) (see Fig. 2C). Why such mutations cause a defect in melanosome transport, has never been elucidated at the molecular level, nor
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