Melanoregulin (Mreg), a product of the dilute suppressor gene, has been implicated in the regulation of melanosome transport in mammalian epidermal melanocytes, given that Mreg deficiency was found to restore peripheral melanosome distribution from perinuclear melanosome aggregation in Rab27A-deficient melanocytes. However, the function of Mreg in melanosome transport has remained unclear. Here, we show that Mreg regulates microtubule-dependent retrograde melanosome transport through the dynein–dynactin motor complex. Mreg interacted with the C-terminal domain of Rab-interacting lysosomal protein (RILP) and formed a complex with RILP and p150Glued (also known as dynactin subunit 1, DCTN1), a component of the dynein–dynactin motor complex, in cultured cells. Overexpression of Mreg, RILP or both, in normal melanocytes induced perinuclear melanosome aggregation, whereas knockdown of Mreg or functional disruption of the dynein–dynactin motor complex restored peripheral melanosome distribution in Rab27A-deficient melanocytes. These findings reveal a new mechanism by which the dynein–dynactin motor complex recognizes Mreg on mature melanosomes through interaction with RILP and is involved in the centripetal movement of melanosomes.

Introduction

Melanosomes are specialized organelles that synthesize and store melanin pigments in pigment cells. Pigmentation of mammalian hair and skin requires proper formation and transport of melanosomes along two cytoskeletal elements, namely microtubules and actin filaments. Unlike other organelles, melanosomes are exceptionally visible under a light microscope, and thus are considered a good model system for analyzing organelle formation and transport (Marks and Seabra, 2001; Raposo and Marks, 2007). Three types of movements by melanosomes have been defined on the basis of their speed, direction and location in the cell: fast anterograde movement towards the plus-ends of microtubules at the periphery of the cell; fast retrograde movement towards the minus-ends of microtubules at the cell center; and slow movement on actin filaments at the cell periphery (Wu et al., 1998).

During the past decade, a tripartite protein complex composed of the small GTPase Rab27A, its specific effector Slac2a (also called melanophilin, MLPH) and the actin-based motor myosin Va, has been shown to mediate actin-based melanosome transport in mammalian epidermal melanocytes (Fukuda et al., 2002; Provance et al., 2002; Strom et al., 2002; Wu et al., 2002; Fukuda, 2005), and the Rab-myosin transport system now appears to be a fundamental mechanism of organelle transport in many cell types (Seabra and Coudrier, 2004; Akhmanova and Hammer, 2010). Disruption of the tripartite protein complex, e.g. upon deficiency of Rab27A, causes the pigmentary dilution in human Griscelli syndrome patients and the corresponding mouse model [(Van Gele et al., 2009) and references therein] as a result of melanosome clustering around the nucleus (i.e. a defect in the transfer of melanosomes from microtubules to actin filaments). Interestingly, the diluted coat color phenotype of dilute (myosin Va-deficient), ashen (Rab27A-deficient) and leaden (Slac2a-deficient) mice was found to be restored by a second mutation in the Dsu (dilute suppressor) locus in 1983 (Sweet, 1983; Moore et al., 1988). However, although the Dsu gene product, melanoregulin (Mreg; also called Dsu) (O'Sullivan et al., 2004; Boesze-Battaglia et al., 2007), has been implicated in the regulation of microtubule-dependent melanosome transport (Marks and Seabra, 2001), whose molecular machinery also remains to be elucidated in mammalian epidermal melanocytes, the function of Mreg in melanosome transport has remained unknown for more than 25 years.

In this study, we investigated the involvement of Mreg in the retrograde melanosome transport in melanocytes that is dependent on the dynein–dynactin motor complex. We showed that Mreg forms a complex with Rab-interacting lysosomal protein (RILP) and p150Glued (also known as dynactin subunit 1, DCTN1), a component of the dynein–dynactin motor complex. A possible function of Mreg as a cargo receptor for the dynein–dynactin motor complex is discussed on the basis of our findings.

Results

Involvement of Mreg in microtubule-dependent retrograde melanosome transport

To investigate the possible role of Mreg in microtubule-dependent melanosome transport, we first overexpressed EGFP-tagged Mreg protein in black-mouse-derived melan-a cells (Bennett et al., 1987). As shown in Fig. 1A, EGFP–Mreg was clearly targeted to mature melanosomes (arrows in the inset of Fig. 1A; see also Fig. 2; endogenous Mreg is localized on mature melanosomes), and ~50% of the Mreg-expressing cells exhibited the perinuclear melanosome aggregation phenotype (Fig. 1E). Although Damek-Poprawa and co-workers have previously reported that Mreg–EGFP is localized to late endosomes and lysosomes, but not to mature melanosomes, in melanocytes (Damek-Poprawa et al., 2009), this discrepancy might be attributable to the use of different Mreg constructs, i.e. EGFP–Mreg (N-terminal EGFP tag in this study) and Mreg–EGFP (C-terminal EGFP tag in the previous study), suggesting that C-terminal EGFP-tagging prevents the melanosomal localization of Mreg. To confirm the melanosomal localization of endogenous Mreg protein, mature melanosomes were affinity-purified using anti-Rab27A IgG and the presence of Mreg in the melanosomal fractions was investigated by immunoblotting. As expected, Mreg clearly co-purified with three melanosome markers, Rab27A, tyrosinase and Tyrp1, but was not co-purified with other organelle markers (Fig. 2).

Fig. 1.

Knockdown of Mreg restores peripheral dispersion of melanosomes in melan-ash cells. (A) Expression of EGFP–Mreg (right), but not EGFP alone (left), induced perinuclear melanosome aggregation in melan-a cells. Note that Mreg strongly colocalized with melanosomes (arrows; melanosomes are pseudo-colored in red in the upper insets of the top panels, and the corresponding bright-field images are shown in the lower insets of the top panels). Bright-field images show the melanosome distribution in the cells (top). EGFP-expressing cells are outlined with a broken red line. The insets show magnified views of the boxed areas. (B) Knockdown of Mreg with specific shRNA had no effect on the peripheral melanosome distribution in melan-a cells. Melan-a cells were transfected with pSilencer-Mreg-st1 or -st2 (expressing Mreg shRNA #1 and #2, respectively) together with pEGFP-C1 and Mreg-deficient cells were identified by GFP fluorescence (bottom panels). (C) Knockdown of Mreg in melan-ash cells with specific shRNAs (or expression of EGFP–Rab27A) caused dispersion of melanosomes from around the nucleus to the cell periphery. Note that the melanosomes in the Mreg-deficient melan-ash cells were not observed just beneath the plasma membrane, in contrast to the EGFP–Rab27A-re-expressing melan-ash cells (i.e. rescued cells). Because the peripheral dispersion phenotype induced by Mreg shRNA #1 in melan-ash cells was completely restored by the coexpression of siRNA-resistant Mreg (supplementary material Fig. S1), the observed effect is not likely to have been caused by an off-target effect of shRNA. Scale bars: 10 μm. (D) RNAi-mediated knockdown of Mreg in melan-a cells (a) and melan-ash cells (b) as revealed by an RT-PCR analysis. G3PDH expression (bottom panels) is shown as a reference to ensure that equivalent amounts of first strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (E,F) The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in A,B. (G) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test).

Fig. 1.

Knockdown of Mreg restores peripheral dispersion of melanosomes in melan-ash cells. (A) Expression of EGFP–Mreg (right), but not EGFP alone (left), induced perinuclear melanosome aggregation in melan-a cells. Note that Mreg strongly colocalized with melanosomes (arrows; melanosomes are pseudo-colored in red in the upper insets of the top panels, and the corresponding bright-field images are shown in the lower insets of the top panels). Bright-field images show the melanosome distribution in the cells (top). EGFP-expressing cells are outlined with a broken red line. The insets show magnified views of the boxed areas. (B) Knockdown of Mreg with specific shRNA had no effect on the peripheral melanosome distribution in melan-a cells. Melan-a cells were transfected with pSilencer-Mreg-st1 or -st2 (expressing Mreg shRNA #1 and #2, respectively) together with pEGFP-C1 and Mreg-deficient cells were identified by GFP fluorescence (bottom panels). (C) Knockdown of Mreg in melan-ash cells with specific shRNAs (or expression of EGFP–Rab27A) caused dispersion of melanosomes from around the nucleus to the cell periphery. Note that the melanosomes in the Mreg-deficient melan-ash cells were not observed just beneath the plasma membrane, in contrast to the EGFP–Rab27A-re-expressing melan-ash cells (i.e. rescued cells). Because the peripheral dispersion phenotype induced by Mreg shRNA #1 in melan-ash cells was completely restored by the coexpression of siRNA-resistant Mreg (supplementary material Fig. S1), the observed effect is not likely to have been caused by an off-target effect of shRNA. Scale bars: 10 μm. (D) RNAi-mediated knockdown of Mreg in melan-a cells (a) and melan-ash cells (b) as revealed by an RT-PCR analysis. G3PDH expression (bottom panels) is shown as a reference to ensure that equivalent amounts of first strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (E,F) The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in A,B. (G) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test).

Fig. 2.

Endogenous Mreg protein is localized to mature melanosomes. Immunoaffinity purification of Rab27A-bound melanosomes from B16-F1 cells with anti-Rab27A-IgG-conjugated magnetic beads was performed as described previously (Kuroda and Fukuda, 2004). Melanosomal fractions were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with anti-Mreg antibody (0.6 μg/ml), anti-Rab27A antibody (1.6 μg/ml), anti-tyrosinase antibody (0.3 μg/ml), anti-Tyrp1 antibody (0.1 μg/ml), anti-Lamp1 antibody (1 μg/ml), anti-EEA1 antibody (1:1000 dilution), anti-TfR antibody (0.5 μg/ml), anti-GM130 antibody (0.3 μg/ml), anti-calreticulin antibody (1:1000 dilution) and anti-γ-adaptin antibody (0.25 μg/ml). The amount of IgG heavy chain (HC) used for immunoprecipitation was determined by Amido Black staining of the blots (bottom panel). Input (lane 1) shows 1% volume of the crude membrane fractions used for immunoaffinity-purification. Note that Mreg was co-purified with melanosome markers (lane 3 in the top four panels), but not other organelle markers. The positions of the molecular mass markers (×10−3) are shown on the left.

Fig. 2.

Endogenous Mreg protein is localized to mature melanosomes. Immunoaffinity purification of Rab27A-bound melanosomes from B16-F1 cells with anti-Rab27A-IgG-conjugated magnetic beads was performed as described previously (Kuroda and Fukuda, 2004). Melanosomal fractions were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with anti-Mreg antibody (0.6 μg/ml), anti-Rab27A antibody (1.6 μg/ml), anti-tyrosinase antibody (0.3 μg/ml), anti-Tyrp1 antibody (0.1 μg/ml), anti-Lamp1 antibody (1 μg/ml), anti-EEA1 antibody (1:1000 dilution), anti-TfR antibody (0.5 μg/ml), anti-GM130 antibody (0.3 μg/ml), anti-calreticulin antibody (1:1000 dilution) and anti-γ-adaptin antibody (0.25 μg/ml). The amount of IgG heavy chain (HC) used for immunoprecipitation was determined by Amido Black staining of the blots (bottom panel). Input (lane 1) shows 1% volume of the crude membrane fractions used for immunoaffinity-purification. Note that Mreg was co-purified with melanosome markers (lane 3 in the top four panels), but not other organelle markers. The positions of the molecular mass markers (×10−3) are shown on the left.

Consistent with a previous report on Mreg-null mutant mice (Moore et al., 1988), knockdown of endogenous Mreg in melan-a cells with specific shRNA had no effect on peripheral melanosome distribution [Fig. 1B,D(a),F], whereas the same shRNA is able to re-disperse melanosomes to the cell periphery in Rab27A-defective melan-ash cells (Ali et al., 2004), which normally exhibit melanosome clustering around the nucleus (Bahadoran et al., 2001; Hume et al., 2001) [Fig. 1C,D(b),G; supplementary material Fig. S1]. It was evident, however, that the melanosomes in the Mreg-deficient melan-ash cells were not attached to the plasma membrane (Fig. 1C, insets in the middle two panels) because of a defect in the Rab27A–Slp2a complex that is required for anchoring of melanosomes to the plasma membrane (Kuroda and Fukuda, 2004), in contrast to normal melan-a cells and Rab27A-re-expressing melan-ash cells, whose peripheral melanosomes were often observed just beneath the plasma membrane (Fig. 1C, inset in the far right panel).

The above results prompted us to hypothesize that Mreg is involved in the regulation of microtubule-dependent retrograde melanosome transport, and to test our hypothesis we investigated the involvement of the dynein–dynactin motor complex in the melanosome aggregation phenotype in melan-ash cells. As expected, disruption of the dynein–dynactin motor complex either by expression of p50dynamitin (also known as dynactin subunit 2, DCTN2), a negative regulator of dynein function (Burkhardt et al., 1997), or knockdown of p150Glued, a subunit of dynactin, in melan-ash cells, phenocopied Mreg-deficient melan-ash cells (i.e. there was re-dispersion of melanosomes to the cell periphery) (Fig. 3A,B,D,E,G). It should be noted that the Mreg-induced perinuclear melanosome aggregation phenotype in melan-a cells was almost completely eliminated by coexpression with p50dynamitin (Fig. 3C,F), strongly suggesting that Mreg is involved in retrograde melanosome transport through regulation of the dynein–dynactin motor complex.

Mreg forms a complex with RILP and p150Glued both in vitro and in melanocytes

The results of a biochemical screening for the Mreg-binding partner in the dynein–dynactin motor complex or among its associated proteins in a coexpression assay in COS-7 cells indicated that RILP (Fig. 4A), one of the p150Glued-binding proteins involved in lysosomal transport to the cell center (Cantalupo et al., 2001; Jordens et al., 2001), interacted with Mreg (Fig. 4B, lane 7 in the middle row). Interestingly, Mreg was unable to interact with RILP-L1, a close homologue of RILP (Wang et al., 2004) (Fig. 4A) (Fig. 4B, lane 8 in the middle row). Consistent with these findings, knockdown of endogenous RILP, but not of RILP-Ll, in melan-ash cells also phenocopied Mreg-deficient melan-ash cells (Fig. 4C–E; supplementary material Fig. S2), but the same RILP shRNA had no effect on the peripheral melanosome distribution in melan-a cells, in the same manner as the Mreg shRNA had no effect (supplementary material Fig. S2D).

Deletion analysis of RILP indicated that Mreg binds the C-terminal half of RILP (called RILP-C), which contains a second coiled-coil domain (CC2) (Fig. 4F, lane 3 in the top panel). We also attempted to determine the location of the RILP-binding site in Mreg and prepared two deletion constructs, ΔN and ΔC (Fig. 5A). Interestingly, we found that RILP binds the C-terminal portion of Mreg, ΔN (Fig. 5B, lane 3), although the C-terminal portion of Mreg is not involved in melanosomal localization (Fig. 5C, middle panels). By contrast, the N-terminal portion of Mreg (ΔC; Fig. 5C, bottom panels) was sufficient to mediate melanosomal localization, although this portion lacked RILP-binding activity (Fig. 5B, lane 4).

Fig. 3.

Functional disruption of a dynein–dynactin motor complex restores the peripheral dispersion of melanosomes in melan-ash cells. (A) Expression of p50dynamitin caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control pEGFP-C1 (left) or pEGFP-C1-p50dynamitin (right). (B) Knockdown of p150Glued with specific siRNAs caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control siRNA or siRNAs targeting p150Glued and immunostained with anti-p150Glued antibody (bottom). Bright-field images show the melanosome distribution in the cells (top). EGFP (or siRNA)-expressing cells are outlined with a broken red line. (C) Coexpression of p50dynamitin with Mreg restored the peripheral melanosome distribution of melan-a cells. Melan-a cells were transfected with a control pEF-T7-GST vector (left) or pEF-T7-p50dynamitin (right) together with pEF-FLAG-Mreg. Bright-field images show the melanosome distribution in the cells (bottom). Scale bars: 10 μm. (D,E) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in A,B. (F) The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes expressing FLAG–Mreg for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test). (G) Efficiency of siRNAs targeted against p150Glued. The siRNAs against p150Glued were transfected into melan-a cells, and their cell lysates were subjected to SDS-PAGE (10% gels) followed by immunoblotting with anti-p150Glued antibody (1:4000 dilution) and anti-actin antibody (1:10,000 dilution). The positions of the molecular mass markers (×10−3) are shown on the left.

Fig. 3.

Functional disruption of a dynein–dynactin motor complex restores the peripheral dispersion of melanosomes in melan-ash cells. (A) Expression of p50dynamitin caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control pEGFP-C1 (left) or pEGFP-C1-p50dynamitin (right). (B) Knockdown of p150Glued with specific siRNAs caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control siRNA or siRNAs targeting p150Glued and immunostained with anti-p150Glued antibody (bottom). Bright-field images show the melanosome distribution in the cells (top). EGFP (or siRNA)-expressing cells are outlined with a broken red line. (C) Coexpression of p50dynamitin with Mreg restored the peripheral melanosome distribution of melan-a cells. Melan-a cells were transfected with a control pEF-T7-GST vector (left) or pEF-T7-p50dynamitin (right) together with pEF-FLAG-Mreg. Bright-field images show the melanosome distribution in the cells (bottom). Scale bars: 10 μm. (D,E) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in A,B. (F) The number of melanocytes showing perinuclear melanosome aggregation is expressed as a percentage of the number of melanocytes expressing FLAG–Mreg for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test). (G) Efficiency of siRNAs targeted against p150Glued. The siRNAs against p150Glued were transfected into melan-a cells, and their cell lysates were subjected to SDS-PAGE (10% gels) followed by immunoblotting with anti-p150Glued antibody (1:4000 dilution) and anti-actin antibody (1:10,000 dilution). The positions of the molecular mass markers (×10−3) are shown on the left.

Because RILP was originally described as a Rab7 effector (Fig. 4B, lane 3 in the middle row) and the C-terminal half of RILP is responsible for Rab7-binding activity (Cantalupo et al., 2001; Wu et al., 2005), we proceeded to explore the relationship between Rab7 binding and Mreg binding of RILP in vitro. A competition assay indicated that Rab7 and Mreg compete with each other for RILP binding (i.e. Mreg binding to RILP was clearly suppressed in the presence of an increasing amount of Rab7; Fig. 4G, compare the top two panels) indicating that Mreg occupies the same or overlapping binding site as Rab7, which does not allow for simultaneous binding. We did not directly compare the affinity of the RILP–Rab7 interaction with that of the RILP–Mreg interaction, but, as judged from the intensity of the bands in (Fig. 4B), Rab7 seemed to bind RILP preferentially (Fig. 4G, compare lanes 3 and 7 in the middle panel).

Although Rab7 has been shown to function as a RILP–p150Glued receptor in lysosomal transport (Jordens et al., 2001), on the basis of the following observations Rab7 itself is unlikely to function in the process of retrograde melanosome transport. First, Rab7 is not present on mature melanosomes (supplementary material Fig. S3A, insets) and has already been shown to be involved in the transport of ‘early stage’ melanosomes and not of mature melanosomes in primary human melanocytes (Jordens et al., 2006). Second, in contrast to Mreg overexpression, overexpression of a dominant-active Rab7 mutant [i.e. Rab7(Q67L)] in melan-a cells had no effect on melanosome distribution (supplementary material Fig. S3A). Third, RNAi-mediated knockdown of Rab7 in melan-ash cells failed to restore peripheral melanosome distribution (supplementary material Fig. S3B–D). To determine whether Mreg functions as a melanosomal receptor for RILP–p150Glued in the retrograde transport of mature melanosomes, by analogy to the function of Rab7 as a receptor for RILP–p150Glued in lysosomal transport, we performed in vitro binding experiments using purified components (see Materials and Methods for details). As shown in Fig. 5D, Mreg formed a complex with purified RILP and p150Glued (lane 2 in the top panel) and Mreg alone did not directly interact with p150Glued (lane 1 in the top panel). Furthermore, the results of the co-immunoprecipitation assays using anti-Mreg-specific antibody indicated that endogenous Mreg is actually associated with p150Glued in cultured melanocytes (Fig. 5E, lane 4 in the second panel; supplementary material Fig. S4). The association between Mreg and p150Glued is likely to be dependent on RILP, because the knockdown of endogenous RILP caused a reduction in the amount of p150Glued that was associated with Mreg (Fig. 5E, compare lane 4 with 5 and 6 in the second and third panels).

Fig. 4.

Mreg interacts with RILP. (A) Schematic representation of the mouse RILP and RILP-L1. The two coiled-coil (CC) domains (named CC1 and CC2) are indicated by shaded boxes. The degree of amino acid identity in the CC domains of RILP and RILP-L1 is indicated by percentages. (B) Interaction between Mreg and RILP, as revealed by a co-immunoprecipitation assay in COS-7 cells. Beads coupled to either FLAG–Rab27A (lanes 1 and 2), FLAG–Rab7 (lanes 3 and 4), FLAG–Rab34 (lanes 5 and 6) and FLAG–Mreg (lanes 7 and 8) were incubated with COS-7 cell lysates containing T7–RILP (lanes 1, 3, 5 and 7) or T7–RILP-L1 (lanes 2, 4, 6 and 8), and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. Note that both RILP and RILP-L1 interacted with Rab34 (lanes 5 and 6 in the middle panel) but that only RILP interacted with Rab7 and Mreg (lanes 3 and 7 in the middle panel). (C) Knockdown of RILP, but not of RILP-L1, with specific shRNAs caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control vector, RILP shRNA expression vectors, or RILP-L1 shRNA expression vectors together with the EGFP expression vector as a transfection marker. Bright-field images show the melanosome distribution in the cells. EGFP-expressing cells are outlined with a broken red line. Because the peripheral dispersion phenotype induced by RILP shRNA #1 in melan-ash cells was completely restored by the coexpression of siRNA-resistant RILP (supplementary material Fig. S2), the observed effect is not likely to have been caused by an off-target effect of the shRNA. Scale bars: 10 μm. (D) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test). (E) RNAi-mediated knockdown of RILP (a) and RILP-L1 (b) in melan-ash cells as revealed by an RT-PCR analysis. G3PDH expression (bottom panels) is shown as a reference to ensure that equivalent amounts of first-strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (F) Mapping of the site responsible for Mreg-binding in RILP. The truncated mutants of RILP used in this study (RILP-N and RILP-C) are shown at the top. Glutathione–Sepharose beads coupled to nothing, T7–GST–RILP-N or T7–GST–RILP-C were incubated with solutions containing HA–Mreg, and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. (G) Competition experiments revealed that Mreg-binding and Rab7-binding of RILP are mutually exclusive. Glutathione–Sepharose beads coupled to T7–GST–RILP-C or beads alone were incubated with solutions containing T7–Mreg and/or increasing amount of FLAG–Rab7, and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. Input shows a 1:80 volume (B,F) or 1:100 volume (G), of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers (×10−3) are shown on the left.

Fig. 4.

Mreg interacts with RILP. (A) Schematic representation of the mouse RILP and RILP-L1. The two coiled-coil (CC) domains (named CC1 and CC2) are indicated by shaded boxes. The degree of amino acid identity in the CC domains of RILP and RILP-L1 is indicated by percentages. (B) Interaction between Mreg and RILP, as revealed by a co-immunoprecipitation assay in COS-7 cells. Beads coupled to either FLAG–Rab27A (lanes 1 and 2), FLAG–Rab7 (lanes 3 and 4), FLAG–Rab34 (lanes 5 and 6) and FLAG–Mreg (lanes 7 and 8) were incubated with COS-7 cell lysates containing T7–RILP (lanes 1, 3, 5 and 7) or T7–RILP-L1 (lanes 2, 4, 6 and 8), and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. Note that both RILP and RILP-L1 interacted with Rab34 (lanes 5 and 6 in the middle panel) but that only RILP interacted with Rab7 and Mreg (lanes 3 and 7 in the middle panel). (C) Knockdown of RILP, but not of RILP-L1, with specific shRNAs caused melanosomes to disperse from around the nucleus to the cell periphery. Melan-ash cells were transfected with a control vector, RILP shRNA expression vectors, or RILP-L1 shRNA expression vectors together with the EGFP expression vector as a transfection marker. Bright-field images show the melanosome distribution in the cells. EGFP-expressing cells are outlined with a broken red line. Because the peripheral dispersion phenotype induced by RILP shRNA #1 in melan-ash cells was completely restored by the coexpression of siRNA-resistant RILP (supplementary material Fig. S2), the observed effect is not likely to have been caused by an off-target effect of the shRNA. Scale bars: 10 μm. (D) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing EGFP fluorescence for the experiments shown in C. *P<0.01 in comparison with the control (Student's unpaired t test). (E) RNAi-mediated knockdown of RILP (a) and RILP-L1 (b) in melan-ash cells as revealed by an RT-PCR analysis. G3PDH expression (bottom panels) is shown as a reference to ensure that equivalent amounts of first-strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (F) Mapping of the site responsible for Mreg-binding in RILP. The truncated mutants of RILP used in this study (RILP-N and RILP-C) are shown at the top. Glutathione–Sepharose beads coupled to nothing, T7–GST–RILP-N or T7–GST–RILP-C were incubated with solutions containing HA–Mreg, and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. (G) Competition experiments revealed that Mreg-binding and Rab7-binding of RILP are mutually exclusive. Glutathione–Sepharose beads coupled to T7–GST–RILP-C or beads alone were incubated with solutions containing T7–Mreg and/or increasing amount of FLAG–Rab7, and the proteins bound to the beads were analyzed by immunoblotting with indicated antibodies. Input shows a 1:80 volume (B,F) or 1:100 volume (G), of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers (×10−3) are shown on the left.

Mreg functions as a RILP–p150Glued receptor for retrograde melanosome transport

If Mreg actually functions as a RILP–p150Glued receptor, expression of the Mreg-binding site of RILP (i.e. RILP-C) in melan-ash cells should cause re-dispersion of the melanosomes by disrupting the endogenous Mreg–RILP interaction (i.e. a dominant-negative effect). As expected, monomeric strawberry (mStr)-tagged RILP-C was targeted to mature melanosomes in melan-ash cells (Fig. 6A, arrows, upper inset in the bottom right panel), and its expression resulted in re-dispersion of the melanosomes to the cell periphery, as occurs upon Mreg knockdown in melan-ash cells (Fig. 6A,B). By contrast, mStr–RILP-N was mostly present in the cytosol and did not rescue the perinuclear melanosome aggregation phenotype in melan-ash cells (Fig. 6A,B). Moreover, coexpression of mStr–RILP-C with EGFP–Mreg in melan-a cells also rescued the perinuclear melanosome aggregation phenotype induced by EGFP–Mreg (Fig. 6C,D), whereas expression of mStr–RILP-C alone in melan-a cells had no effect on peripheral melanosome distribution (supplementary material Fig. S5). By contrast, mStr–RILP-N again had no effect on melanosome distribution in control melan-a cells or EGFP–Mreg-expressing melan-a cells (Fig. 6C,D; supplementary material Fig. S5). In contrast to the truncated RILP mutants, coexpression of full-length RILP with EGFP–Mreg in melan-a cells further increased the proportion of melanocytes showing perinuclear melanosome aggregation to ~80%, a much greater proportion than induced by solo expression of mStr–RILP-full or EGFP–Mreg (~40%) (Fig. 6C,D).

Discussion

In the present study, we have identified RILP as a binding partner of Mreg on mature melanosomes and demonstrated that Mreg is associated with the dynein–dynactin motor complex through interaction with RILP, a p150Glued-binding partner (Fig. 4). In vitro binding experiments using purified components indicated that Mreg is able to directly interact with RILP (Fig. 5D). However, because the in vitro interaction between Mreg and RILP appeared to be weak, we cannot completely rule out the possibility that an additional unidentified factor might stabilize the Mreg–RILP–p150Glued interaction in melanocytes. RNAi-mediated knockdown of Mreg, RILP or p150Glued (or overexpression of p50dynamitin) in melan-ash cells resulted in the same melanosome dispersion phenotype (Figs. 1, 3 and Fig. 4C). This phenomenon is not a unique event in melan-ash cells, because the knockdown of Mreg, RILP or p150Glued in cytochalasin D-treated melan-a cells also caused melanosomes to disperse from around the nucleus to the cell periphery (supplementary material Fig. S6). By contrast, expression of both Mreg and RILP in melan-a cells strongly induced perinuclear aggregation of melanosomes (Fig. 6). These results allowed us to conclude that the Mreg on mature melanosomes [i.e. lysosome-related organelles (Marks and Seabra, 2001; Blott and Griffiths, 2002)] recruits the dynein–dynactin motor complex through RILP, which is analogous to the recruitment of the dynein–dynactin motor complex to lysosomes (or secretory lysosomes) through Rab7 (Jordens et al., 2001; Johansson et al., 2007; Daniele et al., 2011) (a schematic model is shown in supplementary material Fig. S7). In contrast to our finding that the knockdown of Mreg in melan-ash cells restores the peripheral melanosome distribution (Fig. 1C), however, O'Sullivan et al. previously reported that dsu, dilute double mutant did not reverse the dilute phenotype of perinuclear melanosome aggregation (O'Sullivan et al., 2004). This discrepancy might be reconciled by the presence of an alternative compensatory machinery. An additional dynein–dynactin cargo receptor other than Mreg might be present on mature melanosomes. Future extensive study will be necessary to address this issue.

Fig. 5.

Mapping of the sites responsible for RILP binding and melanosomal localization of Mreg, and its interaction with RILP–p150Glued. (A) Schematic representation of the mouse Mreg and its truncated mutants (named Mreg-ΔN and Mreg-ΔC) used in this study. (B) Interaction between RILP and the C-terminal portion of Mreg, as revealed by a co-immunoprecipitation assay in COS-7 cells. Beads coupled to nothing (lane 1), FLAG–Mreg (lane 2), FLAG–Mreg-ΔN (lane 3) or FLAG–Mreg-ΔC (lane 4), were incubated with COS-7 cell lysates containing T7–GST–RILP, and the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with HRP-conjugated anti-T7 tag antibody (top panel) and HRP-conjugated anti-FLAG tag antibody (bottom panel). Input shows a 1:80 volume of the reaction mixture used for immunoprecipitation (middle panel). Note that the C-terminal portion of Mreg (140–214) is necessary for RILP binding. (C) The N-terminal portion of Mreg is required for melanosomal localization in melan-a cells. Melan-a cells were transfected with pEGFP-C1-Mreg (top panels), pEGFP-C1-Mreg-ΔN (middle panels) or pEGFP-C1-Mreg-ΔC (bottom panels). Bright-field images show the melanosome distribution in the cells (right panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in red in the upper inset of the right panels). Note that both Mreg and Mreg-ΔC, but not Mreg-ΔN, colocalized with mature melanosomes. Scale bars: 10 μm. (D) In vitro formation of the Mreg–RILP–p150Glued complex from purified components. Beads coupled to FLAG–p150Glued were incubated with purified GST–Mreg in the absence or presence of T7–GST–RILP, and the proteins bound to the beads were analyzed by immunoblotting with the antibodies indicated. (E) Formation of the Mreg–RILP–p150Glued complex in B16-F1 cells. Endogenous Mreg molecules were immunoprecipitated from lysates of B16-F1 cells expressing control shRNA (lanes 1 and 4) or RILP shRNA #1 or #2 (lanes 2, 3, 5 and 6) with anti-Mreg specific IgG (lanes 4–6) or control IgG (lanes 1–3). Mreg, RILP and p150Glued were analyzed by immunoblotting with specific antibodies. It should be noted that the amount of co-immunoprecipitated p150Glued with Mreg was clearly reduced in RILP-knockdown cells (second panel). Because of the presence of the IgG heavy chain, co-immunoprecipitated RILP was not detected under our experimental conditions (data not shown). However, when FLAG–RILP was expressed in melanocytes, the co-immunoprecipitation of FLAG–RILP with endogenous Mreg and p150Glued was evident (supplementary material Fig. S4), indicating that the Mreg–RILP interaction probably occurs in cultured melanocytes. Input shows a 1:100 volume (D,E), of the reaction mixture used for immunoprecipitation (IP). The positions of the molecular mass markers (×10−3) are shown on the left.

Fig. 5.

Mapping of the sites responsible for RILP binding and melanosomal localization of Mreg, and its interaction with RILP–p150Glued. (A) Schematic representation of the mouse Mreg and its truncated mutants (named Mreg-ΔN and Mreg-ΔC) used in this study. (B) Interaction between RILP and the C-terminal portion of Mreg, as revealed by a co-immunoprecipitation assay in COS-7 cells. Beads coupled to nothing (lane 1), FLAG–Mreg (lane 2), FLAG–Mreg-ΔN (lane 3) or FLAG–Mreg-ΔC (lane 4), were incubated with COS-7 cell lysates containing T7–GST–RILP, and the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with HRP-conjugated anti-T7 tag antibody (top panel) and HRP-conjugated anti-FLAG tag antibody (bottom panel). Input shows a 1:80 volume of the reaction mixture used for immunoprecipitation (middle panel). Note that the C-terminal portion of Mreg (140–214) is necessary for RILP binding. (C) The N-terminal portion of Mreg is required for melanosomal localization in melan-a cells. Melan-a cells were transfected with pEGFP-C1-Mreg (top panels), pEGFP-C1-Mreg-ΔN (middle panels) or pEGFP-C1-Mreg-ΔC (bottom panels). Bright-field images show the melanosome distribution in the cells (right panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in red in the upper inset of the right panels). Note that both Mreg and Mreg-ΔC, but not Mreg-ΔN, colocalized with mature melanosomes. Scale bars: 10 μm. (D) In vitro formation of the Mreg–RILP–p150Glued complex from purified components. Beads coupled to FLAG–p150Glued were incubated with purified GST–Mreg in the absence or presence of T7–GST–RILP, and the proteins bound to the beads were analyzed by immunoblotting with the antibodies indicated. (E) Formation of the Mreg–RILP–p150Glued complex in B16-F1 cells. Endogenous Mreg molecules were immunoprecipitated from lysates of B16-F1 cells expressing control shRNA (lanes 1 and 4) or RILP shRNA #1 or #2 (lanes 2, 3, 5 and 6) with anti-Mreg specific IgG (lanes 4–6) or control IgG (lanes 1–3). Mreg, RILP and p150Glued were analyzed by immunoblotting with specific antibodies. It should be noted that the amount of co-immunoprecipitated p150Glued with Mreg was clearly reduced in RILP-knockdown cells (second panel). Because of the presence of the IgG heavy chain, co-immunoprecipitated RILP was not detected under our experimental conditions (data not shown). However, when FLAG–RILP was expressed in melanocytes, the co-immunoprecipitation of FLAG–RILP with endogenous Mreg and p150Glued was evident (supplementary material Fig. S4), indicating that the Mreg–RILP interaction probably occurs in cultured melanocytes. Input shows a 1:100 volume (D,E), of the reaction mixture used for immunoprecipitation (IP). The positions of the molecular mass markers (×10−3) are shown on the left.

Fig. 6.

Effect of expression of the C-terminal domain of RILP on melanosome distribution in melanocytes. (A) Expression of RILP-C in melan-ash cells restored the peripheral dispersion of melanosomes. Melan-ash cells were transfected with a control pmStr-C1 vector (top panels), pmStr-C1-RILP-N (middle panels) or pmStr-C1-RILP-C (bottom panels). Bright-field images show the melanosome distribution in the cells (right panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in green). mStr-expressing cells are outlined with a broken red line. Note that mStr–RILP-C often colocalized with mature melanosomes (arrows in the upper inset of the bottom right panel; corresponding bright-field image is shown in the lower inset), whereas no melanosomal localization of mStr–RILP-N was observed (red and green signals were clearly separate). Scale bars: 10 μm. (B) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing mStr fluorescence for the experiments shown in A. *P<0.01 in comparison with the control (Student's unpaired t test). The data shown are means+s.d. of data from three independent experiments (n>50). (C) Expression of the C-terminal domain of RILP (RILP-C) in melan-a cells attenuated the perinuclear melanosome aggregation phenotype induced by Mreg expression. Melan-a cells were transfected with a control pmStr-C1 vector (left panels), pmStr-C1-RILP-full (second column panels), pmStr-C1-RILP-N (third column panels) or pmStr-C1-RILP-C (right panels) together with pEGFP-C1-Mreg. Bright-field images show the melanosome distribution in the cells (bottom panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in blue). pmStr-expressing cells are outlined with a broken red line. Note that mStr–RILP-C often colocalized with mature melanosomes together with Mreg (arrows and arrowheads in the insets) and clearly attenuated the perinuclear aggregation phenotype induced by expression of Mreg. Scale bars: 10 μm. (D) The number of melanocytes showing perinuclear aggregation is expressed as a percentage of the number of melanocytes bearing mStr fluorescence for the experiments shown in C (coexpression of EGFP–Mreg and mStr–RILP mutants, right four bars and supplementary material Fig. S5; expression of mStr–RILP mutants alone, left four bars). *P<0.01 in comparison with the mStr-expressing cells or mStr- and EGFP–Mreg-expressing cells (Student's unpaired t test). The data shown are means+s.d. for data from three independent experiments (n>50).

Fig. 6.

Effect of expression of the C-terminal domain of RILP on melanosome distribution in melanocytes. (A) Expression of RILP-C in melan-ash cells restored the peripheral dispersion of melanosomes. Melan-ash cells were transfected with a control pmStr-C1 vector (top panels), pmStr-C1-RILP-N (middle panels) or pmStr-C1-RILP-C (bottom panels). Bright-field images show the melanosome distribution in the cells (right panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in green). mStr-expressing cells are outlined with a broken red line. Note that mStr–RILP-C often colocalized with mature melanosomes (arrows in the upper inset of the bottom right panel; corresponding bright-field image is shown in the lower inset), whereas no melanosomal localization of mStr–RILP-N was observed (red and green signals were clearly separate). Scale bars: 10 μm. (B) The number of melanocytes showing peripheral dispersion is expressed as a percentage of the number of melanocytes bearing mStr fluorescence for the experiments shown in A. *P<0.01 in comparison with the control (Student's unpaired t test). The data shown are means+s.d. of data from three independent experiments (n>50). (C) Expression of the C-terminal domain of RILP (RILP-C) in melan-a cells attenuated the perinuclear melanosome aggregation phenotype induced by Mreg expression. Melan-a cells were transfected with a control pmStr-C1 vector (left panels), pmStr-C1-RILP-full (second column panels), pmStr-C1-RILP-N (third column panels) or pmStr-C1-RILP-C (right panels) together with pEGFP-C1-Mreg. Bright-field images show the melanosome distribution in the cells (bottom panels). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in blue). pmStr-expressing cells are outlined with a broken red line. Note that mStr–RILP-C often colocalized with mature melanosomes together with Mreg (arrows and arrowheads in the insets) and clearly attenuated the perinuclear aggregation phenotype induced by expression of Mreg. Scale bars: 10 μm. (D) The number of melanocytes showing perinuclear aggregation is expressed as a percentage of the number of melanocytes bearing mStr fluorescence for the experiments shown in C (coexpression of EGFP–Mreg and mStr–RILP mutants, right four bars and supplementary material Fig. S5; expression of mStr–RILP mutants alone, left four bars). *P<0.01 in comparison with the mStr-expressing cells or mStr- and EGFP–Mreg-expressing cells (Student's unpaired t test). The data shown are means+s.d. for data from three independent experiments (n>50).

Given that both Mreg and RILP are expressed in a variety of mammalian tissues in addition to melanocytes (O'Sullivan et al., 2004; Wang et al., 2004; Damek-Poprawa et al., 2009), they might also be involved in the retrograde transport of Mreg-containing organelles to the cell center in other cell types. Interestingly, it has recently been reported that lysosome-dependent phagosome degradation activity is diminished in the retinal pigment epithelial cells of Mreg-knockout mice (Damek-Poprawa et al., 2009). If Mreg is present on cathepsin D transport vesicles or engulfed materials in retinal pigment epithelial cells, this phenotype might be explained by inefficient retrograde transport of the vesicles to the cell center, where lysosomes are usually abundant. Further work is needed to determine whether Mreg actually functions as a cargo receptor for RILP–p150Glued in retrograde transport in other cell types, including retinal pigment epithelial cells.

In conclusion, this study is the first to demonstrate that Mreg regulates microtubule-dependent retrograde melanosome transport through regulation of the dynein–dynactin motor complex, probably as a cargo receptor on mature melanosomes. Therefore, functional loss of Mreg in melan-ash cells caused re-distribution of melanosomes from the perinucleus to the cell periphery by suppression of retrograde melanosome transport.

Materials and Methods

Antibodies

The following antibodies used in this study were obtained commercially: anti-actin goat polyclonal antibody and horseradish peroxidase (HRP)-conjugated anti-GST antibody (Santa Cruz Biotechnology); anit-calreticulin rabbit polyclonal antibody (Thermo-Fisher Scientific); anti-EEA1 (early endosomal antigen-1) rabbit monoclonal antibody (Cell Signaling Technology); anti-EGFR (epidermal growth factor receptor) sheep polyclonal antibody (Fitzgerald Industries International, Concord, MA, USA); anti-GM130 mouse monoclonal antibody, anti-γ-adaptin mouse monoclonal antibody, anti-Lamp1 (lysosomal-associated membrane protein 1) rat monoclonal antibody (1D4b), and anti-p150Glued mouse monoclonal antibody (BD Biosciences); anti-Lamp1 rabbit polyclonal antibody (Abcam); anti-Pmel17 mouse monoclonal antibody (HMB45; Dako, Carpinteria, CA, USA); anti-TfR (transferrin receptor) mouse monoclonal antibody (Invitrogen); anti-Tyrp1 (tyrosinase-related protein 1) mouse monoclonal antibody (Ta99; ID Labs, London, ON, Canada); anti-GFP (green fluorescent protein) rabbit polyclonal antibody (MBL, Nagoya, Japan); and HRP-conjugated anti-T7 tag mouse monoclonal antibody (Merck Biosciences Novagen). Anti-tyrosinase rabbit polyclonal antibody and anti-Rab27A rabbit polyclonal antibody were prepared as described previously (Saegusa et al., 2006; Beaumont et al., 2011). Anti-FLAG rabbit polyclonal antibody, HRP-conjugated anti-FLAG tag (M2) mouse monoclonal antibody and anti-FLAG tag antibody-conjugated agaroase were obtained from Sigma-Aldrich.

GST–Mreg, GST–RILP-N (amino acids 1–199), and GST–Rab7 were expressed in bacteria and purified by standard protocols as described previously (Kuroda and Fukuda, 2005). Japanese White rabbits were immunized with the purified GST–Mreg (GST–RILP-N or GST–Rab7), and specific antibodies were affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories) according to the manufacturer's instructions (Fukuda and Mikoshiba, 1999).

Plasmid construction

cDNAs encoding the open reading frame of the mouse Mreg, RILP, p50dynamitin and p150Glued were amplified from Marathon-Ready adult mouse brain, liver and/or testis cDNA (BD Biosciences Clontech) by PCR with specific pairs of oligonucleotides as described previously (Fukuda et al., 1999). Truncated mutants of RILP (i.e. RILP-N, amino acids 1–199; and RILP-C, amino acids 200–369; see Fig. 4F; and of Mreg, i.e. Mreg-ΔN, amino acids 76–214; and Mreg-ΔC, amino acids 1–139; Fig. 5A) were also prepared by conventional PCR techniques. The oligonucleotide sequences used in this study are available from the authors on request. The cDNA fragments amplified were subcloned into the pEGFP-C1 vector (BD Biosciences Clontech), pmStr-C1 vector, which was obtained by replacing the EGFP insert of pEGFP-C1 with mStr, pEF-T7/T7-GST/FLAG/HA vectors (Fukuda et al., 1999), and/or pGEX-4T-3 vector (GE Healthcare). pSilencer 2.1-U6 neo vector was from Ambion. pSilencer-Mreg-st1 (#1) (19-base target site: 5′-CTGCACTGCCTTCCATTTC-3′), pSilencer-Mreg-st2 (#2) (19-base target site: 5′-TCCGTATTCCTCCTTTGGA-3′), pSilencer-RILP-st1 (#1) (19-base target site: 5′-CAGAGCTTGGAACCTGATG-3′), pSilencer-RILP-st2 (#2) (19-base target site: 5′-GTCCAAGGTGTTTCTGCTG-3′), pSilencer-RILP-L1-st1 (#1) (19-base target site: 5′-GAATGAGGACGTCGAGGCT-3′), pSilencer-RILP-L1-st2 (#2) (19-base target site: 5′-GGAGGTGGTGGACAAGCAG-3′), pSilencer-Rab7-st1 (#1) (19-base target site: 5′-TTCCCTGAACCCATCAAAC-3′) and pSilencer-Rab7-st2 (#2) (19-base target site: 5′-GAAAGTGTTGCTGAAGGTC-3′) were constructed essentially as described previously (Kuroda and Fukuda, 2004). Stealth RNA oligonucleotides against mouse p150Glued was obtained from Invitrogen (catalogue numbers MSS203510 and MSS203511; designated as #1 and #2, respectively). Because similar results were obtained with two independent shRNAs and/or siRNAs against Mreg, RILP, RILP-L1, p150Glued or Rab7, the effect of the siRNAs and shRNAs reported in this study are not likely to be an off-target effect. pEGFP-C1-MregSR and pEGFP-C-1-RILPSR (siRNA-resistant mutants) were prepared by two-step PCR techniques as described previously (Tamura et al., 2011). Other expression plasmids, including pEF-FLAG-Rab7, pEF-FLAG-Rab27A, pEF-FLAG-Rab34, pEGFP-C1-Rab7, pEGFP-C1-Rab7(Q67L), and pEF-FLAG-RILP-L1, were prepared as described previously (Fukuda, 2003; Tsuboi and Fukuda, 2006; Fukuda et al., 2008).

Immunofluorescence analysis and melanosome distribution assay

The immortal mouse melanocyte cell lines melan-a, derived from a black mouse, and melan-ash, derived from an ashen mouse (a generous gift of Dorothy C. Bennett, St George's Hospital Medical School, London, UK), were cultured on glass-bottomed dishes (35-mm dish; MatTek, Ashland, MA, USA) as described previously (Bennett et al., 1987; Kuroda et al., 2003; Ali et al., 2004). B16-F1 cells were also cultured as described previously (Kuroda et al., 2002). Plasmids (e.g. pmStr, pEGFP and pSilencer vectors) were transfected into melanocytes by using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Two days after transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and stained with the following antibodies: anti-p150Glued mouse monoclonal antibody (1:100 dilution), anti-Mreg rabbit antibody (18.7 μg/ml), anti-FLAG rabbit polyclonal antibody (1:100 dilution) and anti-T7 tag mouse monoclonal antibody (1:300 dilution). The antibodies were visualized with anti-mouse or rabbit Alexa-Fluor-488 or -594-conjugated IgG (Invitrogen), and examined for fluorescence with a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). The images were processed with Adobe Photoshop software (CS3). Melanosome distribution assays (i.e. perinuclear aggregation versus peripheral dispersion) were performed as described previously (n>50 from three independent dishes) (Kuroda et al., 2003), and data are expressed as means+s.d. Statistical analyses were performed with Student's unpaired t test.

RT-PCR analysis

The total RNA of mouse melan-a cells (or melan-ash cells) that had been transfected with pSilencer vectors twice was prepared using TRI-reagent (Sigma-Aldrich Corp.) and was subjected to reverse transcription (RT) using ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The cDNA encoding mouse Mreg, RILP or RILP-L1 was amplified by PCR with the following pairs of oligonucleotides: 5′-GGATCCAATTTATGGAGCATGCCT-3′ (Mreg-5′ primer, sense) and 5′-GTCGACCATTTGGCTAGCAATCTT-3′ (Mreg-3′ primer, antisense) for Mreg; 5′-TCCAGAAGCTGTCCAGTGTG-3′ (RILP-5′ primer, sense) and 5′-TTGCTGTGGGGTCTCTTCTC-3′ (RILP-3′ primer, antisense) for RILP; and 5′-GGATCCATGGAGGAGCCGCTAGGGTC-3′ (RILP-L1-5′ primer, sense) and 5′-GTCGACCTCCTCCTCCCCATT-3′ (RILP-L1-3′ primer, antisense) for RILP-L1. The mouse G3PDH (glyceraldehyde 3-phosphate dehydrogenase; GAPDH) was amplified using mouse G3PDH-5′ and G3PDH-3′ primers (BD Biosciences Clontech) as a reference.

Immunoaffinity purification of mature melanosomes

Immunoaffinity purification of Rab27A-bound melanosomes with anti-Rab27A-IgG-conjugated magnetic beads was also performed as described previously (Kuroda and Fukuda, 2004). In brief, B16-F1 cells (one confluent 10-cm dish) were homogenized in a homogenization buffer [5 mM HEPES-KOH pH 7.2, 5 mM EGTA, 0.03 M sucrose and protease inhibitors (Complete; Roche Applied Science)]. After centrifugation at 800 g for 10 minutes, the supernatant obtained was incubated for 2 hours at 4°C with anti-Rab27A rabbit polyclonal antibody or control rabbit IgG in the presence of 1% BSA (bovine serum albumin), and then with Dynabeads M-280 (Invitrogen) for 30 minutes at 4°C. After washing the beads twice with PBS (phosphate-buffered saline), the bound fractions were subjected to SDS-PAGE (10% gels) followed by immunoblotting with the antibodies indicated in Fig. 2.

In vitro binding assays

Co-immunoprecipitation assays in COS-7 cells (Fig. 4B; Fig. 5B) and GST pull-down assays (Fig. 4F) were performed as described previously (Fukuda et al., 1999; Fukuda and Kanno, 2005). In brief, T7–GST–RILP-N or T7–GST–RILP-C was transiently expressed in COS-7 cells and affinity-purified by glutathione–Sepharose beads (GE Healthcare). Beads coupled to either T7–GST–RILP-N or T7–RILP-C were incubated for 2 hours at 4°C with COS-7 cell lysates containing HA–Mreg. After washing the beads three times with 1 ml of washing buffer (50 mM HEPES-KOH pH 7.2, 150 mM NaCl, and 1 mM MgCl2), the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with HRP-conjugated anti-T7 tag antibody and anti-Mreg antibody. Similarly, FLAG–Mreg, FLAG–Mreg-ΔN or FLAG–Mreg-ΔC was transiently expressed in COS-7 cells and affinity-purified by anti-FLAG tag antibody-conjugated agarose. Beads coupled to FLAG–Mreg truncated proteins were incubated for 2 hours at 4°C with COS-7 cell lysates containing T7–GST–RILP, and the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-FLAG tag antibody.

Competition experiments between Mreg and Rab7 (Fig. 4G) was performed essentially as described previously (Fukuda et al., 2004). In brief, T7–GST–RILP-C was transiently expressed in COS-7 cells and affinity-purified by glutathione–Sepharose beads. Beads coupled to T7–GST–RILP-C or beads alone were incubated for 2 hours at 4°C with COS-7 cell lysates containing T7–Mreg and/or increasing amount of FLAG–Rab7 in 50 mM HEPES-KOH pH 7.2, 150 mM NaCl, 1 mM MgCl2 and 0.5 mM GTPγS. After washing the beads three times with 1 ml of the washing buffer, the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with anti-Mreg antibody, HRP-conjugated anti-FLAG tag antibody and HRP-conjugated anti-T7 tag antibody.

For in vitro direct-binding experiments, T7–GST–RILP and FLAG–p150Glued were transiently expressed in COS-7 cells and affinity-purified by glutathione–Sepharose beads and anti-FLAG tag-conjugated agarose beads, respectively, as described previously (Fukuda and Kanno, 2005). GST–Mreg was prepared from bacteria as described above. Beads coupled to FLAG–p150Glued were incubated for 2 hours with purified GST–Mreg alone or GST–Mreg and T7–GST–RILP in 50 mM HEPES-KOH pH 7.2, 150 mM NaCl, 1 mM MgCl2 and protease inhibitors (Complete; Roche Applied Science). After washing the beads three times with 1 ml of the washing buffer, the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with HRP-conjugated anti-GST antibody and HRP-conjugated anti-FLAG tag antibody.

Immunoprecipitation

Immunoprecipitation of the endogenous molecules from melanocyte lysates (Fig. 5E; supplementary material Fig. S4) was performed essentially as described previously (Fukuda and Kanno, 2005). In brief, the lysates from B16-F1 cells transfected with pSilencer-RILP-st1 or -st2 (#1 and #2) or pEF-FLAG-RILP were incubated for 2 hours at 4°C with either anti-Mreg rabbit polyclonal antibody described above or control rabbit IgG, and then with protein-A–Sepharose beads for 1 hour at 4°C. After washing the beads three times with 1 ml of washing buffer, the proteins bound to the beads were analyzed by SDS-PAGE (10% gels) followed by immunoblotting with the indicated antibodies in Fig. 5E and supplementary material Fig. S4.

Acknowledgements

We thank Dorothy C. Bennett for kindly donating melan-a and melan-ash cells, Megumi Aizawa for technical assistance and members of the Fukuda Laboratory for valuable discussions.

Funding

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology (MEXT) of Japan (to N.O. and M.F.) and by a grant from the Global COE Program (Basic and Translational Research Center for Global Brain Science) of the MEXT of Japan (to N.O. and M.F.).

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Supplementary information