Functional Analysis of Slac2-c/MyRIP as a Linker Protein between Melanosomes and Myosin VIIa*[boxs]

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c·myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A·Slac2-c·myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Rab27A is a member of the small GTPase Rab family (reviewed in Refs. 1 and 2) and has recently been identified as a critical regulator of various types of membrane trafficking, including vesicle exocytosis in some secretory cells and melanosome transport in melanocytes (reviewed in Refs. [3][4][5]. Eleven effector-type Rab27A-binding proteins have been identified to date: Slac2-a/melanophilin, Slac2-b, Slac2-c/MyRIP, Slp1/JFC1, Slp2-a, Slp3-a, Slp4/granuphilin, Slp5, rabphilin, Noc2, and Munc13-4 (5). The members of the Slp and Slac2 families of these Rab27A-binding proteins contain a common Rab27A-binding domain at the N terminus (referred to as the Slp homology domain (SHD) 1 ) (6 -8), although Slp and Slac2 proteins have different C-terminal domains. Slp1 to -5 contain tandem C2 domains of putative phospholipid or protein interaction sites (9), whereas Slac2-a and Slac2-c contain a myosinbinding domain (MBD) in the middle of the molecule and an actin-binding domain (ABD) at the C terminus (10,11).
The Rab27A in mammalian skin melanocytes is localized on mature melanosomes 2 that contain melanin pigments, and it plays a critical role in melanosome transport that is sequentially mediated by two Rab27A effectors, Slac2-a and Slp2-a. First, Slac2-a mediates the transfer of melanosomes from microtubules to peripheral actin filaments by interacting with Rab27A, myosin Va, and actin (11)(12)(13)(14)(15)(16). Second, Slp2-a links the melanosomes to the plasma membrane through interaction with phosphatidylserine and contributes to the accumulation of melanosomes at the periphery (or dendrites) of the cell (17). These peripheral melanosomes are eventually transferred to adjacent keratinocytes by largely unknown mechanisms (18,19). Loss of any one of the components of the protein complex (Rab27A, Slac2-a, myosin Va, and actin) causes abnormal melanosome accumulation around the nucleus of melanocytes in humans (20 -22) and mice (23-25) (i.e. typical Griscelli syndrome phenotype) as a result of failure of melanosome transfer from the microtubules to actin filaments.
Rab27A is also localized on melanosomes in mammalian retinal pigment epithelium (RPE) cells (26,27), where, in contrast to skin melanocytes, it is thought to regulate melanosome transport independently of the function of Slac2-a, because retinal melanosome distribution is unaffected in leaden mice (i.e. slac2-a-deficient mice) (28), suggesting the presence of a different Rab27A effector(s) in RPE cells. We have recently identified Slac2-c as a homologue of Slac2-a and characterized it as a Rab27A-, myosin Va/VIIa-, and actin-binding protein (10). Slac2-c was independently identified as MyRIP (myosin-VIIa-and Rab-interacting protein) by other groups by yeast two-hybrid screening of a human retina cDNA library using the tail region of myosin VIIa as bait (26). Mutations in MYO7A cause the combination of blindness and deafness seen in human Usher syndrome type 1B and the abnormal distribution of retinal melanosomes and photoreceptor opsin that occurs in shaker-1 mice (29 -34). In addition, melanosomes are absent from the apical processes of the RPE cells of ashen (rab27a-deficient) mice (27,28). Based on these observations, Slac2-c has been proposed to function as a linker protein between myosin VIIa and retinal melanosomes in RPE cells, although involvement of Rab27A⅐Slac2-c⅐myosin VIIa complex in melanosome transport in living cells has never been investigated. Despite its proposed function, Slac2-c has recently been shown to regulate granule exocytosis independently of myosin Va and myosin VIIa in pancreatic ␤ cell lines (35), PC12 cells (36), and parotid acinar cells (37), and thereby the function of Slac2-c as a myosin receptor has been questioned.
To address the question of whether Slac2-c actually acts as a functional myosin receptor, we first investigated the function of the Slac2-c⅐myosin VIIa complex in the transport of Rab27Abound melanosomes in skin melanocytes as a model. Slac2-c had a much higher affinity for myosin VIIa than for myosin Va and colocalized with myosin VIIa on melanosomes. The expression of Slac2-c together with myosin VIIa in Slac2-a-depleted melanocytes restored the normal distribution of melanosomes from perinuclear aggregation, whereas the expression of Slac2-c alone did not. We discuss the possible function of Slac2-FIG. 1. Distinct binding activities of Slac2-c for myosin Va and myosin VIIa. A, schematic representation of the mouse Slac2-c constructs used in B and C. Slac2-c contains an N-terminal Rab27A-binding domain composed of two potential ␣-helical regions (SHD1 and SHD2, closed boxes) and two zinc finger motifs (Zn 2ϩ ), a myosin Va-binding domain in the middle (MBD; shaded box), and a C-terminal ABD (hatched box). The amino acid numbers of each construct are given on both sides. The Slac2-c EA mutant contains replacements of three acidic amino acids with alanines in the MBD. The myosin Va-and myosin VIIa-binding activities of each mutant (Ϫ, ϩ/Ϫ, ϩ, or ϩϩ) are summarized on the right. B and C, T7-Slac2-c, T7-⌬SHD, T7-⌬SHD/⌬ABD, or T7-MBD was expressed in COS-7 cells together with FLAG-myosin Va-tail (B) or FLAG-myosin VIIa-tail (C). The cell lysates were subjected to immunoprecipitation (IP) with anti-T7 tag antibody-conjugated agarose (bottom panels), and co-precipitated FLAG-myosin Va/VIIa-tail was detected with horseradish peroxidase-conjugated anti-FLAG-tag antibody (middle panels). input, one-eightieth volume of the reaction mixture (top panels). The results shown are representative of at least two or three independent experiments. The positions of the molecular mass markers (ϫ 10 Ϫ3 ) are shown on the left. Note that myosin Va and myosin VIIa bound different Slac2-c mutants, indicating different mechanisms for the Slac2-c/myosin Va interaction and Slac2-c/myosin VIIa interaction. c⅐myosin VIIa complex in retinal melanosome transport based on these findings.
In Vitro Binding Assays-In vitro T7-Slac2 and FLAG-myosin binding assays in COS-7 cells were performed as described previously (39). SDS-PAGE and immunoblot analyses with horseradish peroxidaseconjugated anti-FLAG-tag antibody (Sigma) and anti-T7-tag antibody (Novagen, Madison, WI) were also performed as described previously (11,38). The immunoreactive bands were visualized by means of enhanced chemiluminescence systems (Amersham Biosciences). The intensity of the bands on x-ray film was quantified with Lane Analyzer (version 3.0) (ATTO, Tokyo, Japan) as described previously (39). The statistical analyses and curve fitting were performed with the Graph-Pad PRISM computer program (version 4.0). The blots shown in this paper are representative of at least two or three independent experiments.
Melanosome Distribution Assay-Melanosome distribution was assayed as described previously (12,17). In brief, bright field images of melan-a cells transfected with fluorescent vector(s) were obtained at random (more than 50 cells/dish, three independent dishes for each plasmid). Cells in which more than 50% of the melanosomes were located around the nucleus were judged to be "aggregated." Data were expressed as means Ϯ S.D. of three independent experiments. 3 Myosin Va-tail used in this study contains melanocyte-specific exon F.

FIG. 2. Slac2-c bound myosin VIIa with a much higher affinity than it bound myosin Va.
A, recombinant myosin Va-tail was systematically diluted as indicated (top panels), and diluted samples were incubated with beads coupled with T7-Slac2-a (bottom left panels) or T7-Slac2-c (bottom right panels). Proteins bound to the beads were analyzed by 7.5% SDS-PAGE, probed with horseradish peroxidaseconjugated anti-FLAG-tag antibody (middle panels), and visualized by enhanced chemiluminescence. Data shown are representative of three independent experiments. B, immunoreactive bands of myosin Va-tail or myosin VIIa-tail on the x-ray film in A were captured and quantified, and the EC 50 values were calculated with GraphPad PRISM software as described under "Experimental Procedures." Bars, S.E. of three independent experiments. Open circles, Slac2-a/myosin Va-tail interaction; open squares, Slac2-c/myosin Va-tail interaction; closed squares, Slac2-c/myosin VIIa-tail interaction. Note that the affinity of the Slac2c/myosin VIIa interaction (EC 50 ϭ 0.077 Ϯ 0.01) was more than 15 times higher than that of the Slac2-c/myosin Va interaction (EC 50 ϭ 1.3 Ϯ 1.2). siRNA-The siRNA expression vector against mouse Slac2-a (target site 5Ј-GAAGGAAATGGAGACAGTG-3Ј) was prepared as described previously (17), using pSilencer TM 1.0-U6 vector (Ambion, Austin, TX), which expresses short hairpin RNA under the control of the U6 promoter. Subconfluent COS-7 cells seeded on 10-cm dishes were transfected with 2 g of pEGFP-C1-Slac2-a, pEGFP-C1-Slac2-a SR , or pEGFP-C1-Slac2-c together with 2 g of either siRNA expression vector against Slac2-a or a control vector. Cell lysates were prepared as described previously (11,39) and subjected to 7.5% SDS-PAGE. Immunoblotting analysis with anti-GFP rabbit polyclonal antibody (1:1000 dilution; MBL Co., Ltd., Nagoya, Japan) and anti-actin (I-19) goat polyclonal antibody (1:300 dilution; Santa Cruz Biotechnology, Inc.) was performed as described above. Melan-a cells seeded on 35-mm glass bottom dishes were transfected with 1 g each of siRNA expression vector and GFP-tagged protein expression vector and analyzed for melanosome distribution as described above.
In the experiment shown in Fig. 5I, melan-a cells were first transfected with 1 g each of pECFP-C1-Slac2-c and pEYFP-C1-myosin VIIa (or their control vectors) using FuGENE6. One day after transfection, the cells were then transfected with 2 g of a chemically synthesized siRNA against the same site of mouse Slac2-a (5Ј-GAAGGAAAUG-GAGACAGUGdTdT-3Ј, sense; B-Bridge International Inc., Sunnyvale, CA) using X-tremeGENE siRNA transfection reagent (Roche Applied Science) to knock down endogenous Slac2-a molecules. The cells were fixed the next day and analyzed for melanosome distribution as described above.

Slac2-c Preferentially Interacts with Myosin VIIa Rather than Myosin Va in Vitro-Although we and others previously
showed that the tail domain of myosin Va and the tail domain of myosin VIIa interact with the C-terminal region of Slac2-c (10,26,36), the precise myosin-binding domain of Slac2-c has not yet been identified; nor has it been determined whether the two myosins bind the same domain of Slac2-c. We therefore attempted to determine the myosin-binding domain of Slac2-c by a co-transfection assay using COS-7 cells. Consistent with the previous reports, Slac2-c interacted with the tail domain of both myosin Va and myosin VIIa irrespective of the presence of the N-terminal SHD (Fig. 1, B and C, middle panels, lanes 1  and 2). First we tested the myosin binding activity of the putative myosin Va-binding domain of Slac2-c (amino acid residues 409 -602), which corresponds to the previously characterized myosin Va-binding domain of Slac2-a (amino acid residues 241-405) (10,12). Unlike Slac2-a, however, this domain alone was insufficient for the interaction with myosin Va to occur (i.e. weak interaction between T7-MBD and myosin Vatail; Fig. 1B, middle panel, lane 4), suggesting that amino acid residues 409 -602 may be the minimal essential myosin Vabinding domain but that an additional sequence is required for full myosin Va binding. We then tested the myosin Va binding activity of the whole middle domain of Slac2-c (⌬SHD/⌬ABD; amino acid residues 146 -701). To our surprise, however, the ⌬SHD/⌬ABD mutant did not interact with myosin Va-tail at all (Fig. 1B, middle panel, lane 3), indicating that the C-terminal ABD of Slac2-c is required for full interaction with myosin Va. On the other hand, the myosin VIIa-tail preferentially interacted with the ⌬SHD/⌬ABD mutant of Slac2-c rather than the full-length Slac2-c or the Slac2-c-⌬SHD mutant (Fig. 1C, middle panel, lane 3), but the putative MBD alone did not interact with myosin VIIa-tail at all (Fig. 1C, middle panel, lane 4). Additionally, since we previously showed that a conserved acidic cluster in the MBD of Slac2-a is required for myosin Va binding activity (12), we prepared a similar mutant of Slac2-c (referred to as Slac2-c EA ; see Fig. 1A) and tested its myosin Va/VIIa-tail binding activity. In contrast to the reduced inter-action between Slac2-c EA and myosin Va-tail, however, the interaction between Slac2-c EA and myosin VIIa-tail remained intact despite the mutation in the MBD of Slac2-c (Supplemental Fig. 1). These results strongly indicated the existence of different mechanisms for myosin Va and myosin VIIa recognition by Slac2-c.
Next, we compared the relative affinity of Slac2-c for myosin Va and myosin VIIa in an attempt to determine which myosins are preferential ligands of Slac2-c in vivo. Purified T7-tagged Slac2-c coupled with agarose beads was incubated with the various concentrations of either recombinant FLAG-tagged myosin Va-tail or myosin VIIa-tail, and bound myosins were analyzed by immunoblotting as described previously (39). Interestingly, Slac2-c bound myosin VIIa-tail with a much higher affinity than it bound myosin Va-tail (Fig. 2B, closed and open  squares, respectively). Although the Slac2-c/myosin Va-tail interaction was not saturated under our experimental conditions, there was more than 15 times greater affinity in the Slac2-c/ myosin VIIa-tail interaction than in the Slac2-c/myosin Va-tail interaction according to their calculated EC 50 values (0.077 Ϯ 0.01 and 1.3 Ϯ 1.2 (means Ϯ S.E.), respectively). The interaction between Slac2-c and myosin Va-tail was also much weaker than between Slac2-a and myosin Va-tail ( Fig. 2A, middle panels). It should be noted that the binding curve for the Slac2-c/myosin VIIa-tail interaction (closed squares) was almost identical to the curve for the Slac2-a/myosin Va-tail interaction (open circles in Fig. 2B; calculated EC 50 ϭ 0.051 Ϯ 0.006), which has been shown to be physiologically relevant in the melanosome transport in melanocytes (39). We therefore concluded that myosin VIIa is preferred to myosin Va as a binding partner for Slac2-c, at least in vitro.

Recombinant Slac2-c Expressed in Melanocytes Forms a Complex with Endogenous Rab27A and Myosin Va-Although
the results of the in vitro binding experiment described above indicated that myosin Va is not the preferred ligand of Slac2-c, it was still possible that the level of expression of myosin Va in living cells is sufficiently high to bind Slac2-c. To investigate this possibility, we examined the interaction between recombinant Slac2-c and endogenous myosin Va in mouse B16-F1 cells, where hardly any endogenous Slac2-c or myosin VIIa expression was detected under our immunoblotting conditions (data not shown). Purified recombinant T7-Slac2-c (or T7-Salc2-a) coupled with beads was incubated with the B16-F1 cell lysates, and proteins trapped by the beads were analyzed by immunoblotting (see "Experimental Procedures" for details). As shown in Fig. 3B, both endogenous Rab27A and myosin Va were co-purified with recombinant Slac2-c (i.e. formation of a tripartite protein complex between Rab27A, Slac2-c, and myosin Va), although the amount of myosin Va co-purified with Slac2-c was much smaller than the amount co-purified with Slac2-a, consistent with the results shown in Fig. 2. This finding prompted us to further investigate the function of the Rab27A⅐Slac2-c⅐myosin Va complex in melanosome transport in melanocytes.
To do so, we adopted a previously established melanosome distribution assay combined with a dominant negative approach for Salc2-a. The results showed that expression of recombinant Slac2-a lacking Rab27A binding activity (i.e. Slac2a E14A ) inhibited normal melanosome transport and induced perinuclear melanosome aggregation in melan-a cells, presumably by trapping endogenous myosin Va (more than 90% of the transfected cells contained aggregated melanosomes), whereas expression of the wild-type Slac2-a had little effect on melanoperinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing GFP fluorescence. Note that, unlike GFP-Slac2-a E14A , expression of GFP-Slac2-c E14A did not strongly induce perinuclear melanosome aggregation, presumably because of the weak association with myosin Va in vivo. Data are expressed as means Ϯ S.D. of three independent experiments (n Ͼ 150). some distribution (ϳ80% of the transfected cells exhibited the normal peripheral melanosome distribution) (12). Since we had previously identified critical amino acid residues in Slac2-a for binding to Rab27A, and the residues were conserved in Slac2-c (12), we introduced a similar mutation into Slac2-c and produced the Slac2-c E14A mutant (substitution of alanine for glutamate at amino acid position 14). As expected, the E14A mutation selectively abrogated Rab27A binding activity (Fig.  3C, top panel, lane 3). Since the Slac2-c E14A mutant interacted normally with myosin Va (Fig. 3C, middle panel, lane 3), this mutant is an ideal tool for assessing the physiological significance of the Slac2-c/myosin Va interaction in melanosome transport in melanocytes.
When GFP-tagged Slac2-c was expressed in melan-a cells, the distribution of GFP-Slac2-c was quite similar to that of the melanosomes (Fig. 3, D and G). Confocal immunofluorescence microscopic analysis showed that endogenous Rab27A and myosin Va were partly colocalized with GFP-Slac2-c, especially on the melanosomes (Fig. 3, D-G, insets, arrowheads), consistent with the results of the immunoprecipitation experiment (Fig.  3B). Approximately 20% of the GFP-Slac2-c-expressing cells (22.8 Ϯ 8.5%; Fig. 3J) exhibited perinuclear melanosome aggregation, which almost perfectly corresponded to the rate of aggregation induced by wild-type GFP-Slac2-a expression (21.9 Ϯ 7.2%; Fig. 3J) (12). By contrast, expression of the GFP-Slac2-c E14A mutant, which weakly associated with myosin Va and not at all with Rab27A, induced moderate perinuclear melanosome aggregation (47.1 Ϯ 12.9%; Fig. 3, H-J) despite the strong inhibitory effect of the GFP-Slac2-a E14A mutant on melanosome transport (91.3 Ϯ 2.2%; Fig. 3J). The weak effect of the GFP-Slac2-c E14A on melanosome transport is most likely attributable to the weak myosin Va binding activity of Slac2-c (Figs. 2B and 3B) and is not due to the low expression level of GFP-Slac2-c E14A , because the expression levels of GFP-Slac2-c and GFP-Slac2-c E14A were not significantly different (see Supplemental Fig. 2); endogenous myosin Va in the melanocytes preferentially bound to endogenous Slac2-a rather than the GFP-Slac2-c E14A mutant.
Slac2-c Cannot Substitute for Slac2-a in Melanosome Transport in Melan-a Cells-To further determine whether Slac2-c can fully substitute for Slac2-a in melanosome transport, we performed a rescue experiment with combined use of RNA interference technology. As described previously, siRNA against Slac2-a suppressed both forced overexpression of GFP-Slac2-a in COS-7 cells (Fig. 4A, top panel, lane 2) and endogenous expression of Slac2-a in melan-a cells (17), which caused perinuclear melanosome aggregation with a high level of probability (82.2 Ϯ 2.0%, Fig. 4, B, C, and H). To rescue the perinuclear melanosome aggregation induced by the Slac2-a knockdown, we designed an siRNA-resistant Slac2-a cDNA, referred to as Slac2-a SR , in which seven nucleotide substitutions were introduced into the siRNA target site of Slac2-a without altering the amino acid sequence. As expected, expression of GFP-Slac2-a SR was resistant to the Slac2-a siRNA in COS-7 cells (Fig. 4A), and the GFP-Slac2-a SR expressed was properly localized on the melanosomes of the melan-a cells (Fig. 4, D and E,   FIG. 4. Slac2-c 1 and 2), pEGFP-C1-Slac2-a SR (lane 3), or pEGFP-C1-Slac2-c (lanes 4 and 5) together with either siRNA expression vector against Slac2-a (lanes 2, 3, and 5) or a control vector (lanes 1 and 4). Cell lysates were prepared and subjected to 7.5% SDS-PAGE, followed by immunoblotting with anti-GFP antibody (top panel) and anti-actin antibody (control, bottom panel). Note that Slac2-a siRNA suppressed expression of GFP-Slac2-a but not of GFP-Slac2-a SR or GFP-Slac2-c. insets, arrowheads). It should be noted that GFP-Slac2-a SR almost completely rescued the perinuclear melanosome aggregation phenotype induced by the Slac2-a siRNA in melan-a cells (16.7 Ϯ 6.9%, Fig. 4, D, E, and H). Since expression of Slac2-c was also resistant to the Slac2-a siRNA (Fig. 4A, top panel, lane 5), consistent with the specificity of the RNA interference technology, we next investigated whether Slac2-c also has a compensatory effect similar to Slac2-a SR by creating a tripartite protein complex composed of Rab27A, Slac2-c, and myosin Va. However, expression of GFP-Slac2-c with the Slac2-a siRNA still caused aggregation of the melanosomes around the nucleus in almost 90% of the cells (87.6 Ϯ 1.1%; Fig.  4, F-H). These results provided strong evidence that Slac2-c cannot fully compensate the loss of Slac2-a in melanosome transport, presumably because of its weak association with endogenous myosin Va in melanocytes.
Slac2-c Is a Functional Myosin VIIa Receptor in Melanosome Transport-Finally, we investigated the function of the Slac2-c⅐myosin VIIa complex in melanosome transport by ectopically expressing Slac2-c and myosin VIIa in melan-a cells. When YFP-tagged myosin VIIa was expressed in melan-a cells, it was distributed to other punctuate structures in the cytoplasm and not enriched on melanosomes (Fig. 5, A and B), and it had no effect on the peripheral distribution of the melanosomes. Consistent with this, expression of YFP-myosin VIIa alone in Slac2-a-deficient melan-a cells did not rescue the perinuclear melanosome aggregation phenotype (84.1 Ϯ 3.8%; Fig. 4H). It should be noted, however, that co-expression of CFP-Slac2-c induced specific localization of YFP-myosin VIIa on the melanosomes (Fig. 5, C-E, insets, arrowheads), suggesting the formation of a Rab27A⅐Slac2-c⅐myosin VIIa complex on the mela-nosomes. On the other hand, the co-expression of YFP-myosin VIIa-tail with CFP-Slac2-c often induced perinuclear melanosome aggregation (more than 60% of the transfected cells), and the nonfunctional Slac2-c⅐myosin VIIa-tail protein complex was mainly localized in the peripheral actin filaments (Fig. 5, F and  G). To further determine whether the Rab27A⅐Slac2-c⅐myosin VIIa complex is capable of transporting melanosomes in vivo, we attempted to rescue the perinuclear melanosome aggregation phenotype induced by the Slac2-a siRNA by the co-expression of CFP-Slac2-c and YFP-myosin VIIa in melan-a cells. Compared with the Slac2-a-deficient melan-a cells expressing only CFP and YFP and exhibiting a high percentage of perinuclear melanosome aggregation (80.6 Ϯ 7.9%), those expressing CFP-Slac2-c and YFP-myosin VIIa demonstrated a considerably lower percentage of perinuclear melanosome aggregation (39.9 Ϯ 2.7%; Fig. 5I). We therefore concluded that the Slac2-c⅐myosin VIIa complex can substitute for the Slac2-a⅐myosin Va complex in melanosome transport and that Slac2-c is a functional myosin VIIa receptor for Rab27A-bound melanosomes.

DISCUSSION
Although Slac2-c was originally identified as a Rab27A-, myosin Va/VIIa-, and actin-binding protein (10,26), it has recently been shown to control regulated granule exocytosis through interaction with Rab27A and actin, but independently of the function of myosin Va/VIIa, in some secretory cells (35)(36)(37). However, recent studies on the rab27a-deficient ashen mice (27) and myosin VIIa-deficient shaker-1 mice (28)  functional involvement of Slac2-c in melanosome transport had ever been elucidated. In the present study, we investigated the possible function of Slac2-c in melanosome transport for the first time and found that Slac2-c expressed in melan-a cells supports melanosome transport through an interaction with co-expressed myosin VIIa but not through an interaction with endogenous myosin Va or co-expressed myosin VIIa-tail (Figs. 4 and 5). Since the interaction between Slac2-c and myosin VIIa is as strong as the interaction between Slac2-a and myosin Va (Fig. 2B) and the Rab27A⅐Slac2-c⅐myosin VIIa complex actually forms on skin melanosomes in living cells, Slac2-c should function as an organelle receptor for myosin VIIa, the same as Slac2-a functions as a myosin Va receptor. In the absence of Slac2-c, myosin VIIa cannot be recruited to the melanosomes in melan-a cells, because Slac2-a interacts with myosin Va but not myosin VIIa (10), and thus endogenous Slac2-a cannot substitute for Slac2-c.
Since the affinity of Slac2-c for myosin Va was much lower than that of Slac2-a, it is questionable whether the Slac2-c/ myosin Va interaction occurs under physiological conditions. The formation of the Slac2-c⅐myosin Va complex in melan-a cells seemed to be limited for the following reasons. First, endogenous myosin Va was co-purified with Slac2-c-conjugated beads, but to a lesser extent than with the Slac2-a-conjugated beads (Fig. 3B). Second, the GFP-Slac2-c expressed in melanocytes was colocalized with myosin Va at the light microscopic level (Fig. 3, D-G), but this must be mainly attributable to simultaneous localization of GFP-Slac2-c with Slac2-a-linked myosin Va on a single melanosome, rather than a direct interaction between GFP-Slac2-c and myosin Va. Third, expression of GFP-Slac2-c E14A caused perinuclear melanosome aggregation in only ϳ50% of the transfected cells (Fig. 3J), whereas a similar mutation in Slac2-a E14A strongly induced perinuclear melanosome aggregation (Ͼ90% of the GFP-Slac2-a E14A -expressing cells). Fourth, Slac2-c did not rescue the Slac2-adeficient phenotype (i.e. perinuclear melanosome aggregation) (Fig. 4). We therefore conclude that Slac2-c alone cannot fully substitute for Slac2-a, at least in melan-a cells, presumably due to the insufficient concentration of endogenous myosin Va. Although myosin Va is also expressed on RPE melanosomes (28) (see Ref. 27; contradictory results were reported), the contribution of myosin Va to retinal melanosome transport must be limited, because abnormal RPE melanosome distribution was observed in shaker-1 mice, in which myosin Va is intact. At this stage, however, we cannot completely rule out the possibility that Slac2-c forms a complex with myosin Va and regulates other organelle transport in other cell types, where endogenous myosin Va (with exon F) expression levels are very high.
In conclusion, we analyzed the molecular interaction between Slac2-c and two unconventional myosins, Va and VIIa, and the in vivo function of the protein complexes they form by using skin melanocytes as a model. Based on our observations, together with previous findings on the function of Slac2-c in secretory cells (35)(36)(37), we propose that Slac2-c possesses two cell type-specific functions: as a linker protein between Rab27A and myosin VIIa in retinal melanosome transport in RPE cells and as a linker protein between secretory granules and actin filaments via the C-terminal ABD, independently of the function of myosin Va/VIIa.