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Originally published In Press as doi:10.1074/jbc.M501465200 on May 30, 2005

J. Biol. Chem., Vol. 280, Issue 30, 28015-28022, July 29, 2005
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Functional Analysis of Slac2-c/MyRIP as a Linker Protein between Melanosomes and Myosin VIIa*

Taruho S. Kuroda{ddagger} and Mitsunori Fukuda§

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

Received for publication, February 8, 2005 , and in revised form, May 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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. 3-5). 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 myosin-binding 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 melanosomes2 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 (11-16). 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 Rab27A·Slac2-c·myosin 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 {beta} 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.



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  		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 {alpha}-helical regions (SHD1 and SHD2, closed boxes) and two zinc finger motifs (Zn2+), 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-cEA mutant contains replacements of three acidic amino acids with alanines in the MBD. The myosin Va- and myosin VIIa-binding activities of each mutant (-, +/-, +, or ++) are summarized on the right. B and C, T7-Slac2-c, T7-{Delta}SHD, T7-{Delta}SHD/{Delta}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 (x 10-3) 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.

 
To address the question of whether Slac2-c actually acts as a functional myosin receptor, we first investigated the function of the Slac2-c·myosin VIIa complex in the transport of Rab27A-bound 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-c·myosin VIIa complex in retinal melanosome transport based on these findings.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—Deletion mutants of Slac2-c (pEF-T7-Slac2-c-{Delta}SHD/{Delta}ABD and -MBD) were constructed by conventional PCR using the following oligonucleotides with a BamHI site (underlined) or a stop codon (in boldface type): 5'-CGGATCCCGTCTGGAGAGCGGTGCCTG-3' ({Delta}SHD primer, sense), 5'-TCACAGGTACACGTTTTCTTCCA-3' ({Delta}ABD primer, antisense), 5'-CGGATCCTTGAGTAAGCTGTGCCCACC-3' (MBD-5' primer, sense), and 5'-CTACTGGTCCTCCCCAGAAG-3' (MBD-3' primer, antisense). Site-directed mutagenesis and construction of Slac2-c point mutants (pEF-T7-Slac2-cE14A and pEF-T7-Slac2-cEA) and small interfering RNA (siRNA)-resistant Slac2-a mutant (pEGFP-C1-Slac2-aSR) were also performed by PCR as described previously (38) using the following oligonucleotides with a restriction enzyme site (underlined) and substituted nucleotides (in boldface type): 5'-GGATCCATGGGGAGGAAGCTGGACCTGTCGGGTCTGACCGACGATGCGAC-3' (E14A primer, sense), 5'-GAATTCCCGGTCCAGCACGGCCTCAGTAGCGGCGAAGCT-3' (EA primer, sense), and 5'-AAGCTTGGAGGAGGGTAACGGTGATAGAGAGCAGACTGATGA-3' (SR primer, sense). Other pEF expression vectors (pEF-FLAG-Rab27A, pEF-T7-Slac2-a, pEF-T7-Slac2-c, pEF-T7-Slac2-c-{Delta}SHD, pEF-FLAG-MC-myosin Va-tail (melanocyte-type),3 and pEF-FLAG-myosin VIIa-tail) were prepared as described previously (7, 10). Construction of pEGFP-C1-Slac2-c, pECFP-C1-Slac2-c, and pEYFP-C1-myosin VIIa was performed by transfer of cDNA inserts from the above pEF vectors with appropriate restriction enzymes.

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 peroxidase-conjugated 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 GraphPad PRISM computer program (version 4.0). The blots shown in this paper are representative of at least two or three independent experiments.

Recombinant T7-Slac2-c (or T7-Slac2-a) expressed in COS-7 cells was affinity-purified with anti-T7 tag antibody-conjugated agarose (Novagen) as described previously (7). B16-F1 cells (one confluent 10-cm dish) were harvested and homogenized in a buffer containing 1 ml of 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 0.5 mM GTP{gamma}S, and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A) in a glass-Teflon Potter homogenizer by 10 strokes at 900-1000 rpm, and the proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removing insoluble material by centrifugation at 15,000 rpm for 10 min, the supernatant was incubated with the above agarose beads coupled with T7-Slac2-a or T7-Slac2-c (wet volume 20 µl) for 1 h at 4 °C. After washing the beads five times with 10 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 0.2% Triton X-100, and protease inhibitors, proteins trapped by the beads were subjected to 10% SDS-PAGE, followed by immunoblotting with anti-Rab27A mouse monoclonal antibody (BD Transduction Laboratories, Lexington, KY), anti-myosin Va rabbit polyclonal antibody (11), and horseradish peroxidase-conjugated anti-T7 tag antibody.

Cell Culture, Transfections, and Immunofluorescence Analysis—Melan-a cells, an immortal black mouse-derived melanocyte cell line (40), were cultured in RPMI 1640 medium (Sigma) supplemented with 2.7 mM HCl, 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, 7.5 µg/ml phenol red, and 0.1 mM 2-mercaptoethanol at 37 °C under 10% CO2. Immediately prior to use, 200 nM phorbol 12-myristate 13-acetate (Sigma) was added to the medium. COS-7 cells and B16-F1 cells were cultured at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. FuGENE6 (1.5 µl/µg plasmid DNA; Roche Applied Science) and Lipofectamine PLUS reagents (Invitrogen) were used for transfection into melan-a and COS-7 cells, respectively, according to the manufacturers' instructions. For plasmid transfection and microscopic analysis, melan-a cells (7.5 x 104 cells, the day before transfection) were seeded on 35-mm glass bottom dishes (MatTek Corp.), and the following amounts of plasmids were transfected into melan-a cells: 2 µg of green fluorescent protein (GFP) expression vector (see Fig. 3, D-J) or 1 µg each of cyan fluorescent protein (CFP) expression vector, and yellow fluorescent protein (YFP) expression vector (see Fig. 5, A-I). At 48-72 h after transfection, cells were fixed with 4% paraformaldehyde (catalogue number 168-20955; Wako Pure Chemicals, Osaka, Japan) for 20 min. For immunostaining of myosin Va and Rab27A, melan-a cells were permeabilized with 0.3% Triton-X-100 for 2 min and blocked with blocking buffer (1% bovine serum albumin and 0.1% Triton-X-100 in phosphate-buffered saline) for 1 h. The cells were then immunostained with anti-myosin Va antibody (1:100 dilution) and anti-Rab27A antibody (1:50 dilution), followed by Alexa Fluor 568 and 633 secondary IgG, respectively (1:5000 dilution; Molecular Probes, Inc., Eugene, OR). Fluorescence and bright field images were acquired and pseudocolored with a confocal laser-scanning microscope (Fluoview; OLYMPUS, Tokyo, Japan), and the images were processed with Adobe Photoshop software (version 7.0).



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  		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 peroxidase-conjugated 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 EC50 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 Slac2-c/myosin VIIa interaction (EC50 = 0.077 ± 0.01) was more than 15 times higher than that of the Slac2-c/myosin Va interaction (EC50 = 1.3 ± 1.2).

 
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 ± S.D. of three independent experiments.

siRNA—The siRNA expression vector against mouse Slac2-a (target site 5'-GAAGGAAATGGAGACAGTG-3') was prepared as described previously (17), using pSilencerTM 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 µg of pEGFP-C1-Slac2-a, pEGFP-C1-Slac2-aSR, or pEGFP-C1-Slac2-c together with 2 µg 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 µg each of siRNA expression vector and GFP-tagged protein expression vector and analyzed for melanosome distribution as described above.



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  		FIG. 3.
Recombinant Slac2-c forms a complex with endogenous Rab27A and myosin Va in melanocytes. A, schematic representation of the Slac2-cE14A mutant. Substitution of alanine for glutamate at amino acid position 14 (E14A) in the SHD1 of Slac2-c causes loss of Rab27A binding activity without affecting myosin Va binding activity (see also lane 3 in C). B, agarose beads coupled with T7-Slac2-a (lane 2) or T7-Slac2-c (lane 3) or beads alone (negative control; lane 4) were incubated with B16-F1 cell lysate, and proteins trapped by the beads were subjected to 7.5% SDS-PAGE, followed by immunoblotting with anti-Rab27A mouse monoclonal antibody (top panel), anti-myosin Va rabbit polyclonal antibody (middle panel), and horseradish peroxidase-conjugated anti-T7 tag antibody (bottom panel). input, one-eightieth volume of the reaction mixture (lane 1). C, agarose beads coupled with T7-Slac2-c (lane 2) or T7-Slac2-cE14A (lane 3) or beads alone (negative control; lane 4) were incubated with B16-F1 cell lysate, and proteins trapped by the beads were subjected to 10% SDS-PAGE, followed by immunoblotting as described in B. input, one-eightieth volume of the reaction mixture (lane 1). The results shown are representative of two independent experiments. The positions of the molecular mass markers (x 10-3) are shown on the left. Due to the difference in the percentage of SDS-polyacrylamide gel, the bands of T7-Slac2-c in B are broader than in C. D-I, melan-a cells transfected with pEGFP-C1-Slac2-c (D-G) or pEGFP-C1-Slac2-aE14A (H and I) were fixed, permeabilized, and immunostained with anti-Rab27A mouse monoclonal antibody and anti-myosin Va rabbit polyclonal antibody, followed by visualization with Alexa Fluor secondary antibodies. Fluorescence and bright field images were acquired with a confocal laser scan microscope. D and H, fluorescence of GFP; E, fluorescence of Rab27A; F, fluorescence of myosin Va, G and I, bright field images of D and H, respectively. The insets show 2-fold magnified views of the boxed area, and the arrowheads indicate individual melanosomes with GFP-Slac2-c, Rab27A, and myosin Va. The cell showing perinuclear melanosome aggregation has been outlined in yellow (I). The bars in D and H represent 10 µm. J, effect of Slac2-c expression on melanosome distribution in melanocytes. The melanosome distribution of melan-a cells expressing the GFP fusion protein indicated was classified as "perinuclear aggregation" or "normal," as described under "Experimental Procedures." The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing GFP fluorescence. Note that, unlike GFP-Slac2-aE14A, expression of GFP-Slac2-cE14A did not strongly induce perinuclear melanosome aggregation, presumably because of the weak association with myosin Va in vivo. Data are expressed as means ± S.D. of three independent experiments (n > 150).

 
In the experiment shown in Fig. 5I, melan-a cells were first transfected with 1 µg 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 µg of a chemically synthesized siRNA against the same site of mouse Slac2-a (5'-GAAGGAAAUGGAGACAGUGdTdT-3', 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 Va-binding 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 ({Delta}SHD/{Delta}ABD; amino acid residues 146-701). To our surprise, however, the {Delta}SHD/{Delta}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 {Delta}SHD/{Delta}ABD mutant of Slac2-c rather than the full-length Slac2-c or the Slac2-c-{Delta}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-cEA; see Fig. 1A) and tested its myosin Va/VIIa-tail binding activity. In contrast to the reduced interaction between Slac2-cEA and myosin Va-tail, however, the interaction between Slac2-cEA 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 EC50 values (0.077 ± 0.01 and 1.3 ± 1.2 (means ± 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 EC50 = 0.051 ± 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 Rab27A·Slac2-c·myosin 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. Slac2-aE14A) 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 melanosome distribution (~80% 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-cE14A 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-cE14A 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.



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  		FIG. 4.
Slac2-c ectopically expressed in melanocytes does not compensate for the Slac2-a deficiency in melanosome transport. A, effect of Slac2-a siRNA on the expression of Slac2-a (lane 2), siRNA-resistant Slac2-a (Slac2-aSR) (lane 3), and Slac2-c (lane 5). COS-7 cells were transfected with pEGFP-C1-Slac2-a (lanes 1 and 2), pEGFP-C1-Slac2-aSR (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-aSR or GFP-Slac2-c. B-H, GFP (B and C), GFP-Slac2-aSR (D and E), GFP-Slac2-c (F and G), or YFP-myosin VIIa together with the Slac2-a siRNA was introduced into melan-a cells. Fluorescence of GFP (B, D, and F) and the corresponding bright field images (C, E, and G, respectively) of the cells were acquired with a confocal laser scan microscope. The insets show 3-fold magnified views of the boxed area, and the arrowheads indicate the localization of GFP-Slac2-aSR on melanosomes (D and E). The cells showing perinuclear melanosome aggregation have been outlined in yellow (C and G). The bars in B, D, and F represent 10 µm. The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing GFP fluorescence (H). Data are expressed as means ± S.D. of three independent experiments (n > 150). Note that expression of GFP-Slac2-aSR, but not of GFP-Slac2-c or YFP-myosin VIIa, restored the defect of melanosome transport induced by the Slac2-a siRNA.

 
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 ± 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 ± 7.2%; Fig. 3J) (12). By contrast, expression of the GFP-Slac2-cE14A mutant, which weakly associated with myosin Va and not at all with Rab27A, induced moderate perinuclear melanosome aggregation (47.1 ± 12.9%; Fig. 3, H-J) despite the strong inhibitory effect of the GFP-Slac2-aE14A mutant on melanosome transport (91.3 ± 2.2%; Fig. 3J). The weak effect of the GFP-Slac2-cE14A 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-cE14A, because the expression levels of GFP-Slac2-c and GFP-Slac2-cE14A 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-cE14A 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 ± 2.0%, Fig. 4, B, C, and H). To rescue the perinuclear melanosome aggregation induced by the Slac2-a knock-down, we designed an siRNA-resistant Slac2-a cDNA, referred to as Slac2-aSR, 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-aSR was resistant to the Slac2-a siRNA in COS-7 cells (Fig. 4A), and the GFP-Slac2-aSR expressed was properly localized on the melanosomes of the melan-a cells (Fig. 4, D and E, insets, arrowheads). It should be noted that GFP-Slac2-aSR almost completely rescued the perinuclear melanosome aggregation phenotype induced by the Slac2-a siRNA in melan-a cells (16.7 ± 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-aSR 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 ± 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.



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  		FIG. 5.
Melanosome transport through Rab27A·Slac2-c·myosin VIIa complex. A-H, YFP-myosin VIIa alone (A and B), CFP-Slac2-c together with YFP-myosin VIIa (C-E), or CFP-Slac2-c together with YFP-myosin VIIa-tail (F-H) was expressed in melan-a cells. CFP/YFP fluorescence and the corresponding bright field images of the cells were acquired with a confocal laser scan microscope. The insets show 3-fold magnified views of the boxed area (A-E). The arrowheads in A and B indicate the absence of myosin VIIa from melanosomes, whereas those in C-E indicate accumulation of Slac2-c and myosin VIIa on melanosomes. The cell showing perinuclear melanosome aggregation has been outlined in yellow (H). Bars in A, C, and F, 10 µm. I, melanosome distribution in the Slac2-a-depleted melan-a cells expressing CFP-Slac2-c and YFP-myosin VIIa. Melan-a cells were transfected with pECFP-C1-Slac2-c and pEYFP-C1-myosin VIIa or empty vectors, followed by treatment with siRNA directed against Slac2-a. The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing CFP and YFP fluorescence. Data are expressed as means ± S.D. of three independent experiments (n > 100). Note that simultaneous expression of CFP-Slac2-c and YFP-myosin VIIa considerably, but not fully, rescued the perinuclear melanosome aggregation phenotype induced by the siRNA directed against Slac2-a.

 
Slac2-c Is a Functional Myosin VIIa Receptor in Melanosome Transport—Finally, we investigated the function of the Slac2-c·myosin 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 ± 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 Rab27A·Slac2-c·myosin VIIa complex on the melanosomes. 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-c·myosin VIIa-tail protein complex was mainly localized in the peripheral actin filaments (Fig. 5, F and G). To further determine whether the Rab27A·Slac2-c·myosin 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 ± 7.9%), those expressing CFP-Slac2-c and YFP-myosin VIIa demonstrated a considerably lower percentage of perinuclear melanosome aggregation (39.9 ± 2.7%; Fig. 5I). We therefore concluded that the Slac2-c·myosin VIIa complex can substitute for the Slac2-a·myosin Va complex in melanosome transport and that Slac2-c is a functional myosin VIIa receptor for Rab27A-bound melanosomes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (35-37). However, recent studies on the rab27a-deficient ashen mice (27) and myosin VIIa-deficient shaker-1 mice (28) have demonstrated a crucial role of Rab27A and myosin VIIa in retinal melanosome distribution in RPE cells and suggested involvement of Slac2-c as a myosin VIIa receptor, although no 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 Rab27A·Slac2-c·myosin 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-c·myosin 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-cE14A caused perinuclear melanosome aggregation in only ~50% of the transfected cells (Fig. 3J), whereas a similar mutation in Slac2-aE14A strongly induced perinuclear melanosome aggregation (>90% of the GFP-Slac2-aE14A-expressing cells). Fourth, Slac2-c did not rescue the Slac2-a-deficient 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 (35-37), 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.


   FOOTNOTES
 
* This work was supported in part by Ministry of Education, Culture, Sports, and Technology of Japan Grants 15689006, 16044248, 17024065, and 17657067 (to M. F.), by the Cosmetology Research Foundation (to M. F.), and by the SHISEIDO Grants for Scientific Research (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains two additional figures. Back

{ddagger} Supported by the Special Postdoctoral Researchers Program of RIKEN. Back

§ To whom correspondence should be addressed: Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-462-4994; Fax: 81-48-462-4995; E-mail: mnfukuda{at}brain.riken.go.jp.

1 The abbreviations used are: SHD, Slp homology domain; ABD, actin-binding domain; CFP, cyan fluorescent protein; GFP, green fluorescent protein; MBD, myosin-binding domain; RPE, retinal pigment epithelium; siRNA, small interfering RNA; YFP, yellow fluorescent protein; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back

2 Unless otherwise stated, the term "melanosomes" is used to represent mammalian skin melanosomes throughout. Back

3 Myosin Va-tail used in this study contains melanocyte-specific exon F. Back


   ACKNOWLEDGMENTS
 
We thank Eiko Kanno and Yukie Ogata for expert technical assistance.



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 DISCUSSION
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