Slac2-a/Melanophilin Contains Multiple PEST-like Sequences That Are Highly Sensitive to Proteolysis

Abstract

The synaptotagmin-like protein homologue lacking C2 domains-a (Slac2-a)/melanophilin was recently identified as the “missing link” between the small GTPase Rab27A and the actin-based motor protein myosin Va. Although formation of a tripartite protein complex by three molecules had been shown to be required for proper melanosome distribution in melanocytes (Kuroda, T. S., Ariga, H., and Fukuda, M. (2003) Mol. Cell. Biol. 23, 5245-5255), the regulatory mechanisms of the complex (i.e. assembly and disassembly of the complex) had never been elucidated. In this study, we discovered that Slac2-a and a closely related isoform, Slac2-c/MyRIP, contain multiple PEST-like sequences (potential signals for rapid protein degradation) in the myosin Va- and actin-binding domains at the C terminus. We found that the C-terminal domain of Slac2-a is highly sensitive to low concentrations of proteases, such as trypsin and calpain, in vitro, whereas the N-terminal Rab27A-binding domain is highly resistant to these proteases. We further found that endogenous calpains selectively cleave Slac2-a, but not Rab27A or myosin Va, in melanocytes. A mutant Slac2-a lacking one of the PEST-like sequences located at the interface between the myosin Va- and actin-binding domains (ΔPEST; amino acids 399-405) is more stable than the wild-type protein, both in vitro and in melanocytes. Expression of the mutant Slac2-a-ΔPEST with an N-terminal green fluorescence protein tag often induced perinuclear aggregation of melanosomes (∼40% of the transfected cells) compared with the wild-type Slac2-a. Our findings suggest that protein degradation of Slac2-a is an essential process for proper melanosome distribution in melanocytes. The synaptotagmin-like protein homologue lacking C2 domains-a (Slac2-a)/melanophilin was recently identified as the “missing link” between the small GTPase Rab27A and the actin-based motor protein myosin Va. Although formation of a tripartite protein complex by three molecules had been shown to be required for proper melanosome distribution in melanocytes (Kuroda, T. S., Ariga, H., and Fukuda, M. (2003) Mol. Cell. Biol. 23, 5245-5255), the regulatory mechanisms of the complex (i.e. assembly and disassembly of the complex) had never been elucidated. In this study, we discovered that Slac2-a and a closely related isoform, Slac2-c/MyRIP, contain multiple PEST-like sequences (potential signals for rapid protein degradation) in the myosin Va- and actin-binding domains at the C terminus. We found that the C-terminal domain of Slac2-a is highly sensitive to low concentrations of proteases, such as trypsin and calpain, in vitro, whereas the N-terminal Rab27A-binding domain is highly resistant to these proteases. We further found that endogenous calpains selectively cleave Slac2-a, but not Rab27A or myosin Va, in melanocytes. A mutant Slac2-a lacking one of the PEST-like sequences located at the interface between the myosin Va- and actin-binding domains (ΔPEST; amino acids 399-405) is more stable than the wild-type protein, both in vitro and in melanocytes. Expression of the mutant Slac2-a-ΔPEST with an N-terminal green fluorescence protein tag often induced perinuclear aggregation of melanosomes (∼40% of the transfected cells) compared with the wild-type Slac2-a. Our findings suggest that protein degradation of Slac2-a is an essential process for proper melanosome distribution in melanocytes. Melanosomes are the specialized organelles that produce and store melanin pigments and are responsible for the pigmentation of mammalian hair and skin. Mature melanosomes are transported from the cell body of melanocytes to the tips of their dendrites by two distinct motors (i.e. a microtubule-dependent motor and an actin-dependent movement). Finally, melanosomes are translocated from the dendrites of the melanocytes into adjacent epidermal keratinocytes (reviewed in Refs. 1Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (349) Google Scholar and 2Jimbow K. Park J.S. Kato F. Hirosaki K. Toyofuku K. Hua C. Yamashita T. Pigm. 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The results of recent genetic analyses of these mutant animals and biochemical studies of their gene products have indicated that a tripartite protein complex formed by myosin Va (dilute gene product), Slac2-a/melanophilin (GS3/leaden gene product), 1The abbreviations used are: Slp(s), synaptotagmin-like protein(s); ABD, actin-binding domain; GFP, green fluorescence protein; HA, hemagglutinin; HRP, horseradish peroxidase; MBD, myosin Va-binding domain; SHD, Slp homology domain. 1The abbreviations used are: Slp(s), synaptotagmin-like protein(s); ABD, actin-binding domain; GFP, green fluorescence protein; HA, hemagglutinin; HRP, horseradish peroxidase; MBD, myosin Va-binding domain; SHD, Slp homology domain. and Rab27A (ashen gene product) is essential for melanosome transfer from the perinuclear region of melanocytes to their actin-rich cell periphery (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. 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Science. 2001; 293: 1317-1320Crossref PubMed Scopus (120) Google Scholar), or certain GTPase-activating protein for Rab27A may be involved in this process through conversion of GTP-Rab27A to GDP-Rab27A.In this study, we discovered that Slac2-a contains multiple PEST-like sequences in the C-terminal domain that are known to be sensitive to proteases such as calpains (33Dice J.F. FASEB J. 1987; 1: 349-357Crossref PubMed Scopus (196) Google Scholar, 34Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1396) Google Scholar) and we found that Slac2-a, but not Rab27A or myosin Va, is selectively degraded by endogenous calpains in melanocytes. We also found that expression of green fluorescence protein (GFP)-tagged Slac2-a lacking one of the PEST-like sequences (ΔPEST) often caused perinuclear aggregation of melanosomes in melan-a cells. Based on our findings, together with the fact that yeast Vac17p, a linker protein of the yeast class V myosin Myo2p contains a functional PEST sequence (35Tang F. Kauffman E.J. Novak J.L. Nau J.J. Catlett N.L. Weisman L.S. Nature. 2003; 422: 87-92Crossref PubMed Scopus (99) Google Scholar, 36Ishikawa K. Catlett N.L. Novak J.L. Tang F. Nau J.J. Weisman L.S. J. Cell Biol. 2003; 160: 887-897Crossref PubMed Scopus (86) Google Scholar), we discuss the convergent evolution of the cargo receptor for class V myosins in membrane trafficking.MATERIALS AND METHODSMaterials—μ-Calpain, calpastatin peptide, and calpain inhibitor III were obtained from Calbiochem-Novachem Corp. Trypsin, thrombin, and horseradish peroxidase (HRP)-conjugated anti-FLAG tag (M2) mouse monoclonal antibody were obtained form Sigma Chemical Co. (St. Louis, MO). HRP-conjugated anti-hemagglutinin (HA) tag and HRP-conjugated anti-T7 tag mouse monoclonal antibodies were from Roche Molecular Biochemicals and Novagen (Madison, WI), respectively. Anti-actin goat polyclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Texas Red-conjugated phalloidin was from Molecular Probes Inc. (Eugene, OR). Anti-Slac2-a-SHD, anti-Slac2-a-ABD, anti-Rab27A, and anti-myosin Va rabbit polyclonal antibodies were prepared as described previously (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 37Fukuda M. Kanno E. Yamamoto A. J. Biol. Chem. 2004; 279: 13065-13075Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 38Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (93) Google Scholar).Plasmid Construction—Addition of the FLAG tag sequence to the C terminus of T7-Slac2-a was essentially performed by PCR as described previously (39Fukuda M. Ibata K. Mikoshiba K. J. Neurochem. 2001; 77: 730-740Crossref PubMed Scopus (26) Google Scholar). A mutant Slac2-a lacking a PEST-like sequence (amino acids 399-405; named ΔPEST) was produced by two-step PCR techniques by using the following mutagenic oligonucleotides having an artificial KpnI site (underlined) as described previously (40Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar): 5′-GGTACCATCTGGCTTGGTGGAACCACTGATGTTGCTGG-3′ (ΔPEST primer 1; antisense) and 5′-GGTACCTTCCTTGGAGGGTC-3′ (ΔPEST primer 2; sense). The mutant Slac2-a-ΔPEST fragment was then subcloned into the modified pEF-T7 expression vector (41Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 42Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) (named pEF-T7-Slac2-a-ΔPEST) or the pEGFP-C1 vector (BD Biosciences Clontech) (named pEGFP-C1-Slac2-a-ΔPEST). Other expression constructs (pEF-T7-Slp3-a, pEF-T7-Slac2-c, pEF-HA-Rab27A, and pEF-FLAG-myosin Va-tail) were produced as described previously (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 23Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The myosin Va-tail used in this study contains a melanocyte-specific exon F (28Seperack P.K. Mercer J.A. Strobel M.C. Copeland N.G. Jenkins N.A. EMBO J. 1995; 14: 2326-2332Crossref PubMed Scopus (114) Google Scholar, 29Huang J.D. Mermall V. Strobel M.C. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. Genetics. 1998; 148: 1963-1972Crossref PubMed Google Scholar).Limited Proteolysis of Slac2 and Slp3-a by Trypsin and Thrombin— Recombinant T7-tagged Slac2 (or Slp3-a) protein was expressed in COS-7 cells and purified with the anti-T7 tag antibody-conjugated agarose (Novagen) essentially as described previously (42Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), but all procedures were performed in the absence of any protease inhibitors. Purified proteins were incubated at 25 °C for 30 min with various concentrations of trypsin (see Fig. 1A) or 1 unit of thrombin in 50 mm HEPES-KOH, pH 7.2. Digested proteins were then analyzed by 10% SDS-PAGE, followed by immunoblotting with HRP-conjugated anti-T7 tag (or anti-FLAG tag) antibody as described previously (43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar).Digestion of Slac2-a by Calpains—Recombinant T7-Slac2-a-FLAG protein was incubated at 30 °C for 15 min with various concentrations of μ-calpain in the presence or absence of 750 μm Ca2+ (Fig. 3, A and B). Digested proteins were then analyzed by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-T7 tag (or anti-FLAG tag) antibody (43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar).Fig. 3Recombinant Slac2-a and Slac2-c are cleaved by μ-calpain in vitro. After incubation of T7-Slac2-a-FLAG in 50 mm HEPES-KOH, pH 7.2, in the presence or absence of 750 μm Ca2+ with the indicated concentrations of μ-calpain at 30 °C for 15 min, the reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (A) or HRP-conjugated anti-FLAG tag antibody (B). Note that the C-terminal domain of Slac2-a was also rapidly degraded by μ-calpain, the same as trypsin. The positions of the three major tryptic fragments of Slac2-a (see Fig. 1A) are indicated as open arrowheads. C, cleavage of recombinant T7-Slac2-c by μ-calpain only in the presence of Ca2+ (lane 2). The N-terminal domain of Slac2-c containing the SHD was also resistant to 50 nm calpain treatment, the same as Slac2-a (arrowhead in C). The positions of the molecular mass markers (×10-3) are shown on the left.View Large Image Figure ViewerDownload (PPT)Melan-a cells (one 10-cm dish at confluence) were homogenized in 500 μl of a buffer (50 mm HEPES-KOH, pH 7.2, 1 mm MgCl2, and 150 mm NaCl) without any protease inhibitors in a glass-Teflon Potter homogenizer with 10 strokes at 1000 rpm, and proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removing the insoluble material by centrifugation at 15,000 rpm for 10 min, cell lysates were incubated at 30 °C for 15 min, 30 min, or 1 h with either 2 mm EGTA or 750 μm Ca2+ in the presence or absence of calpain inhibitors (10 μm calpastatin peptide or 10 μm calpain inhibitor III). Reactions were terminated by adding SDS sample buffer and boiling for 3 min. Protein expression levels of Rab27A, Salc2-a, myosin Va, and actin were analyzed by immunoblotting with specific antibodies as described previously (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The intensity of the bands on x-ray films was quantified with Lane Analyzer (version 3.0) (ATTO, Tokyo, Japan).Miscellaneous Procedures—The immortalized melanocyte cell line melan-a (generous gift of Dorothy C. Bennett) was cultured as described previously (17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (103) Google Scholar, 44Bennett D.C. Cooper P.J. Hart I.R. Int. J. Cancer. 1987; 39: 414-418Crossref PubMed Scopus (396) Google Scholar). Transfection of pEGFP-C1-Slac2-a into melan-a cells with FuGENE 6 (Roche Molecular Biochemicals), immunocytochemistry with Texas-Red phalloidin, and the melanosome aggregation assay were performed as described previously (17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (103) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). The cells were examined for fluorescence and by bright-field images with a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). The melanosome distribution of the transfected cells (“dispersed” indicates a normal peripheral distribution of melanosomes (Fig. 6A, top right); “aggregated” means accumulation of melanosomes in the perinuclear regions (Fig. 6A, bottom right) was evaluated as described previously (17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (103) Google Scholar). Plasmids were transfected into COS-7 cells (7.5 × 105 cells/10 cm-dish, the day before transfection) with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's notes. T7-Slac2-a proteins were immunoprecipitated with anti-T7 tag antibody-conjugated agarose as described previously (42Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). SDS-PAGE and immunoblotting analyses with HRP-conjugated anti-FLAG tag, anti-HA tag, anti-actin, and anti-T7 tag antibodies were also performed as described previously (17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (103) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 43Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The blots shown in this article are representative of at least two or three independent experiments.Fig. 6Deletion of the PEST-like sequence around site 3 induces melanosome aggregation in wild-type melanocytes.A, melan-a cells were transfected with a vector encoding GFP-tagged wild-type Slac2-a (top and middle rows) or mutant GFP-Slac2-a-ΔPEST (bottom row). After cells were fixed and permeabilized with 0.3% Triton X-100, they were stained with Texas Red-conjugated phalloidin. The cells were examined by confocal microscopy for fluorescence of GFP-Slac2-a proteins (green in left column) and actin (red in middle column). Bright-field images (right column) show the melanosome distribution in the cells. Note that the mutant GFP-Slac2-a-ΔPEST or the wild-type protein (with high expression levels) colocalized with actin (yellow in inset of the bottom row, center, or middle row, center, respectively) and induced perinuclear aggregation of melanosomes, whereas the wild-type Slac2-a protein (with low expression levels) was present on melanosomes rather than on actin filaments (top row, middle, inset). Scale bar, 10 μm. B, summary of the results of the melanosome distribution assay. Images of transfected cells were captured at random by using GFP fluorescence as a marker, and we judged whether melanosomes had aggregated in the perinucleus by examining the corresponding bright-field images as described previously (17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (103) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). The results are expressed as percentages of cells exhibiting perinuclear melanosome aggregation, and the values are means ± S.D. of data from three independent experiments (n > 150). *, p < 0.05 (Student's unpaired t test). C, the expression level of GFP-Slac2-a-ΔPEST protein (closed arrowhead) in melan-a cells was twice as great as that of GFP-Slac2-a protein (open arrowhead), even when the same amount of plasmids were used for transfection. Total cell lysates of the transfected cells were analyzed by 7.5% SDS-PAGE and immunoblotting with anti-Slac2-a-SHD (2 μg/ml; top) or anti-actin antibody (1/300 dilution; bottom). The arrow indicates endogenous Slac2-a molecules, whose expression was unaltered by overexpression of GFP-Slac2-a mutants or GFP alone (data not shown). D, mutant GFP-Slac2-a-ΔPEST protein is more resistant to endogenous calpain than wild-type GFP-Slac2-a protein. Total cell lysates of melan-a cells expressing GFP-Slac2-a mutants were prepared in the absence of protease inhibitors as described under “Materials and Methods.” After incubation of the cell lysates in 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, and 1 mm MgCl2 in the presence or absence of 750 μm Ca2+, the reactions were stopped by addition of SDS sample buffer and boiling for 3 min. Samples were then analyzed by 7.5% SDS-PAGE and immunoblotting with anti-Slac2-a-SHD antibody (2 μg/ml). The results shown are representative of two independent experiments. The positions of the molecular mass markers (×10-3) are shown on the left.View Large Image Figure ViewerDownload (PPT)RESULTSSlac2-a Is Highly Sensitive to Proteases—In a previous study, we found that both recombinant Slac2-a and Slac2-c proteins usually contained a variety of degradation products when expressed in cultured cells (27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) (Figs. 1A, lane 1, and 3C, lane 1). Because the recombinant Slp1-5 proteins were expressed and purified with little contamination by degradation products (Fig. 1C, lane 1, and Refs. 23Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar and 24Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. Biochem. Biophys. Res. Commun. 2002; 293: 899-906Crossref PubMed Scopus (63) Google Scholar), Slac2-a proteins should be less stable than Slp proteins in living cells and more sensitive to proteases, and we performed limited proteolysis experiments with trypsin to test this hypothesis (25Fukuda M. J. Biol. Chem. 2002; 277: 40118-40124Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 45Fukuda M. Yamamoto A. Mikoshiba K. J. Biol. Chem. 2001; 276: 41112-41119Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As shown in Fig. 1, A and B, recombinant T7-Slac2-a-FLAG protein was easily digested with 100 ng/ml trypsin at 25 °C for 30 min. To our surprise, however, monitoring the degradation of T7-Slac2-a-FLAG protein with anti-T7 tag antibody (i.e. detection of the N-terminal domain of Slac2-a) yielded three major tryptic fragments (Fig. 1A, sites 1-3, open arrowheads), whereas monitoring the degradation of T7-Slac2-a-FLAG protein with anti-FLAG tag antibody (i.e. detection of the C-terminal domain of Slac2-a) yielded only a single band corresponding to the full-length protein (Fig. 1B, lanes 1-4). The recombinant Slp3-a protein, on the other hand, was highly resistant to trypsin (up to 250 ng/ml). Similar results were obtained when the anti-Slac2-a-SHD and the anti-Slac2-a-actin-binding domain (ABD) antibodies were used to detect the N-terminal and C-terminal domains, respectively, of Slac2-a (data not shown). These results indicated that the C-terminal ABD was highly sensit