Slac2-a/Melanophilin Contains Multiple PEST-like Sequences That Are Highly Sensitive to Proteolysis (2004)

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 (360) Google Scholar and 2Jimbow K. Park J.S. Kato F. Hirosaki K. Toyofuku K. Hua C. Yamashita T. Pigm. Cell Res. 2000; 13: 222-229Crossref PubMed Scopus (110) Google Scholar). Melanosome transfer from the microtubles to actin filaments must be a crucial step for melanosome transport in melanocytes, because defects in this step cause pigment dilution in the skin and hair in human diseases (e.g. Griscelli syndrome) and the corresponding coat-color mutant mice (e.g. dilute, ashen, and leaden) (1Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (360) Google Scholar, 3Pastural E. Barrat F.J. Dufourcq-Lagelouse R. Certain S. Sanal O. Jabado N. Seger R. Griscelli C. Fischer A. de Saint Basile G. Nat. Genet. 1997; 16: 289-292Crossref PubMed Scopus (369) Google Scholar, 4Ménasché G. Pastural E. Feldmann J. Certain S. Ersoy F. Dupuis S. Wulffraat N. Bianchi D. Fischer A. Le Deist F. de Saint Basile G. Nat. Genet. 2000; 25: 173-176Crossref PubMed Scopus (760) Google Scholar, 5Ménasché G. Ho C.H. Sanal O. Feldmann J. Tezcan I. Ersoy F. Houdusse A. Fischer A. de Saint Basile G. J. Clin. Investig. 2003; 112: 450-456Crossref PubMed Scopus (260) Google Scholar, 6Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Nature. 1991; 349: 709-713Crossref PubMed Scopus (468) Google Scholar, 7Wilson S.M. Yip R. Swing D.A. O'Sullivan T.N. Zhang Y. Novak E.K. Swank R.T. Russell L.B. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7933-7938Crossref PubMed Scopus (343) Google Scholar, 8Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (212) Google Scholar). 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. 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. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 10Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (385) Google Scholar, 11Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 12Hume A.N. Collinson L.M. Hopkins C.R. Strom M. Barral D.C. Bossi G. Griffiths G.M. Seabra M.C. Traffic. 2002; 3: 193-202Crossref PubMed Scopus (133) Google Scholar, 13Provance Jr., D.W. James T.L. Mercer J.A. Traffic. 2002; 3: 124-132Crossref PubMed Scopus (113) Google Scholar, 14Nagashima K. Torii S. Yi Z. Igarashi M. Okamoto K. Takeuchi T. Izumi T. FEBS Lett. 2002; 517: 233-238Crossref PubMed Scopus (120) Google Scholar, 15Ménasché G. Feldmann J. Houdusse A. Desaymard C. Fischer A. Goud B. de Saint Basile G. Blood. 2003; 101: 2736-2742Crossref PubMed Scopus (84) Google Scholar, 16Bahadoran P. Busca R. Chiaverini C. Westbroek W. Lambert J. Bille K. Valony G. Fukuda M. Naeyaert J.M. Ortonne J.P. Ballotti R. J. Biol. Chem. 2003; 278: 11386-11392Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar; reviewed in Refs. 19Hammer III, J.A. Wu X.S. Curr. Opin. Cell Biol. 2002; 14: 69-75Crossref PubMed Scopus (125) Google Scholar and 20Fukuda M. Recent Res. Dev. Neurochem. 2002; 5: 297-309Google Scholar). Slac2-a functions as a linker protein between myosin Va and Rab27A and directly and simultaneously binds the GTP-bound form of Rab27A on the melanosome via the N-terminal synaptotagmin-like protein (Slp) 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. homology domain (referred to as SHD or RBD27 (Rab binding domain specific for Rab27 isoforms)) (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 10Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (385) Google Scholar, 11Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Nagashima K. Torii S. Yi Z. Igarashi M. Okamoto K. Takeuchi T. Izumi T. FEBS Lett. 2002; 517: 233-238Crossref PubMed Scopus (120) Google Scholar, 17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 21Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (81) Google Scholar, 22Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (69) 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 (176) Google Scholar, 24Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. Biochem. Biophys. Res. Commun. 2002; 293: 899-906Crossref PubMed Scopus (65) Google Scholar, 25Fukuda M. J. Biol. Chem. 2002; 277: 40118-40124Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Fukuda M. J. Biol. Chem. 2003; 278: 15373-15380Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and myosin Va, an actin-based motor protein, via the large C-terminal domain (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 10Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (385) Google Scholar, 11Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Nagashima K. Torii S. Yi Z. Igarashi M. Okamoto K. Takeuchi T. Izumi T. FEBS Lett. 2002; 517: 233-238Crossref PubMed Scopus (120) Google Scholar, 17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The interaction between Slac2-a and myosin Va is strengthened by the presence of a melanocyte-specific exon F in the tail domain of myosin Va (18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28Seperack P.K. Mercer J.A. Strobel M.C. Copeland N.G. Jenkins N.A. EMBO J. 14: PubMed Scopus Google Scholar, Strobel M.C. Russell L.B. M.S. Copeland N.G. Jenkins N.A. PubMed Google Scholar, Wang F. Rao K. Sellers J.R. Hammer III, J.A. Mol. Biol. Cell. 2002; 13: PubMed Scopus Google Scholar, Cell 2002; PubMed Scopus Google Scholar). Although the formation of a tripartite protein complex is to be essential for melanosome transfer from to actin filaments in mammalian melanocytes, is the of disassembly of the complex the transfer of melanosomes to actin of myosin Va be in from melanosomes as been shown in F. 2001; 293: PubMed Scopus (120) Google or protein for Rab27A be in this process of to In this study, we discovered that Slac2-a multiple PEST-like sequences in the C-terminal domain that are to be sensitive to such as calpains J. PubMed Scopus Google Scholar, M. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar) and we found that Slac2-a, but not Rab27A or myosin Va, is selectively by endogenous calpains in melanocytes. We found that of green fluorescence protein Slac2-a lacking one of the PEST-like sequences often perinuclear aggregation of melanosomes in on with the that a linker protein of the myosin a F. Novak Nature. 2003; PubMed Scopus Google Scholar, K. Novak F. J. Cell Biol. 2003; PubMed Scopus Google we the of the for in and from and horseradish tag form tag and tag from and was from was from and Va as (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, M. E. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. T. H. Fukuda M. J. Cell Sci. 2004; 117: PubMed Scopus Google Scholar). of the tag to the C of was by as M. K. Mikoshiba K. J. Neurochem. 2001; PubMed Scopus Google Scholar). A mutant Slac2-a lacking a PEST-like acids was by by the an as M. T. J. M. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and The mutant Slac2-a-ΔPEST was into the M. J. M. S. Mikoshiba K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Mikoshiba K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (65) Google Scholar, M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) or the and as (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) 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 (176) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The myosin used in this a melanocyte-specific exon F P.K. Mercer J.A. Strobel M.C. Copeland N.G. Jenkins N.A. EMBO J. 14: PubMed Scopus Google Scholar, Strobel M.C. Russell L.B. M.S. Copeland N.G. Jenkins N.A. PubMed Google Scholar). of and by and protein was in and with the tag as M. Mikoshiba K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (65) Google Scholar, M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google but in the of at for with concentrations of trypsin or of in by by with tag as M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Slac2-a by protein was at for with concentrations of in the presence or of A and by by with tag M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). at in of a and in a with at and with at for the by at for cell at for or with or in the presence or of or by and for of myosin Va, and actin by with specific as (9Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The of the on was with cell of C. was as T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, D.C. J. PubMed Scopus Google Scholar). of into with with and the melanosome aggregation as T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). The for fluorescence and by with a fluorescence The melanosome distribution of the transfected a distribution of melanosomes of melanosomes in the perinuclear was as T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar). transfected into the with to the with tag as M. Mikoshiba K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (65) Google Scholar, M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and analyses with and tag as T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, M. E. Mikoshiba K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The shown in this are of at two or three Slac2-a to a study, we found that both Slac2-a and a of degradation products in M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and the and with by degradation products 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 (176) Google Scholar and 24Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. Biochem. Biophys. Res. Commun. 2002; 293: 899-906Crossref PubMed Scopus (65) Google Slac2-a be stable than Slp in and more sensitive to proteases, and we with trypsin to this M. J. Biol. Chem. 2002; 277: 40118-40124Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, M. A. Mikoshiba K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in A and protein was with trypsin at for the degradation of protein with tag (i.e. of the N-terminal domain of three whereas the degradation of protein with tag (i.e. of the C-terminal domain of a corresponding to the protein The protein, on the was highly resistant to trypsin to results the and the domain used to the N-terminal and C-terminal of Slac2-a not results indicated that the C-terminal was highly sensitive to and by whereas the N-terminal SHD of Slac2-a was highly was that the protein was highly sensitive to and the of with was to the with we three with the tag and signals with the tag in A and that both trypsin and cleave the in Slac2-a. The on the was highly resistant to Slac2-a PEST-like by in results of the indicated that Slac2-a protein contain specific sequences that are highly sensitive to proteases. a between Slac2-a and with we found that the C-terminal domain of Slac2-a (i.e. myosin Va-binding domain and multiple PEST-like sequences (potential signals for rapid protein degradation) J. PubMed Scopus Google Scholar, M. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar) that of amino and A and be that the and for more than of the of amino acids in the and that the be the the to of the in the SHD, a that is with the that the SHD is highly resistant to the used in this sequences are often by calpains (reviewed in Refs. J. PubMed Scopus Google Scholar, M. Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar, and H. W. J. Rev. 2003; PubMed Scopus Google we the of on the of Slac2-a in vitro A and the C-terminal domain of in a was to the C-terminal domain of Slac2-a, whereas a of was required for degradation of the N-terminal domain was by in the presence of of the Slac2-a by in further Slac2-a protein is a of endogenous calpains in melanocytes, we cell of the cell at for or in the presence or of endogenous Slac2-a protein was in the presence of and with the results of the and we corresponding to the N-terminal domain of Slac2-a (i.e. is highly resistant to the of the of myosin Va, and actin was by with for and and the tail domain of myosin Va is to contain a Jr., D.W. M.S. Mercer J.A. Biophys. 2000; PubMed Scopus Google Scholar). In we not in the protein of the cell with the of Slac2-a protein as a of to was by to two (i.e. and we that endogenous calpains selectively and cleave the Slac2-a in and and Expression of of Melanosomes in the three we on for further for the was highly sensitive to both trypsin and and to be than because and located the MBD, of the or to myosin Va-binding as shown in the myosin (i.e. or T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). because was located at the interface between the and ABD, of the (ΔPEST; and was not to myosin Va- or actin-binding the mutant Slac2-a-ΔPEST with myosin and the as the wild-type protein and The results of the that the mutant Slac2-a-ΔPEST protein was more resistant to trypsin than the wild-type protein and The mutant Slac2-a-ΔPEST protein was by PEST-like adjacent to and are Finally, we the of of the on melanosome transport in melanocytes. of the of the PEST-like the was as as that of the wild-type protein and and was resistant to compared with the wild-type protein and In of protein induced aggregation of melanosomes in a of (∼40% of the transfected cells) than the wild-type protein of the transfected cells) is that or the wild-type Slac2-a protein more with actin filaments than with melanosomes and in to the wild-type low was on melanosomes than with actin filaments (i.e. green and signals We and had shown that formation of a tripartite protein complex of Slac2-a, and myosin Va is for melanosome transport in melanocytes in X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (385) Google Scholar, 11Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 17Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). the of the disassembly of the complex melanosome transport had been Although of the myosin Va tail been to be in from melanosomes in F. 2001; 293: PubMed Scopus (120) Google the form of myosin Va the binds Slac2-a in vitro (18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google that of the tail of myosin Va is not in the disassembly of the tripartite protein A protein for Rab27A the complex by Rab27A (i.e. of to to of Slac2-a from specific protein been In the study, we discovered that the C-terminal domain of Slac2-a multiple PEST-like sequences and we have of that these sequences are highly sensitive to in vitro and in melanocytes. We that Slac2-a, but not is highly sensitive to proteases, in vitro and and that the Slac2-a in melanocytes is by calpains We that of one of the PEST-like sequences the of mutant Slac2-a protein and that of in melanocytes melanosome aggregation in the at a than the wild-type protein the Slac2-a-ΔPEST mutant the complex with myosin Va, and actin the of of Slac2-a-ΔPEST on melanosome transport be from that of the of the or (i.e. formation of an complex and of melanosome aggregation in of the transfected cells) T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 18Fukuda M. Kuroda T.S. J. Cell Sci. 2004; 117: 583-591Crossref PubMed Scopus (42) Google Scholar). Our results suggest that protein degradation of the Slac2-a is an essential process for melanosome transport in melanocytes. that is melanosome transport be by of wild-type Slac2-a In of the often perinuclear aggregation of and this was than in the Although the of of Slac2-a on actin filaments melanosome transport is we that Slac2-a formation of actin in the cell periphery via the C-terminal ABD, melanosome transfer from to actin because of to actin and in T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (109) Google Scholar, 27Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). a recently been shown to contain a and was that degradation of be in proper transport from cell to cell F. Novak Nature. 2003; PubMed Scopus Google Scholar). Although mammalian Slac2-a and these two myosin simultaneously binds and and formation of the complex is required for transport in K. Novak F. J. Cell Biol. 2003; PubMed Scopus Google the of the Va complex in melanosome transport in both and Slac2-a contain PEST-like sequences that are highly sensitive to K. Novak F. J. Cell Biol. 2003; PubMed Scopus Google Scholar and this of the PEST-like of or Slac2-a their in F. Novak Nature. 2003; PubMed Scopus Google Scholar and this is highly that degradation of the Slac2-a (i.e. disassembly of the transport complex) is in the proper distribution of melanosomes in melanocytes, is the degradation in melanocytes, and not in the degradation been discovered endogenous calpains in melanocytes selectively and the Slac2-a and are that is the in cell H. W. J. Rev. 2003; PubMed Scopus Google Scholar, Cell Res. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, H. H. K. K. J. Biol. Chem. Full Text PDF PubMed Google the be responsible for the degradation of the Slac2-a in with the to endogenous the induced perinuclear aggregation of melanosomes and of the Slac2-a not such be to the specific degradation of endogenous Slac2-a by because we of essential of melanosome transport and not this not a of is to by the is to is in the degradation of Slac2-a in and melanosome was identified as the of Slac2-a and to be in melanosome transport in pigment M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, A. J.S. P. S. C. J.P. U. F. C. EMBO 2002; 3: PubMed Scopus Google Scholar). been shown to from T. Fukuda M. M. A. C. R. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar, C. J.S. S. A. G. I. C. G. de Saint Basile G. C. S. J.P. F. J. Cell Biol. 2003; PubMed Scopus Google Scholar) and A. S. T. H. Fukuda M. J. Cell Sci. 2004; 117: PubMed Scopus Google of the C-terminal domain of PEST-like sequences degradation of protein with this we recently found that protein is selectively and by endogenous proteases, Fukuda, in We that the of in melanosome transport or be by In we have discovered that Slac2-a PEST-like sequences that are highly sensitive to and we further that of one of the PEST-like sequences the protein and perinuclear aggregation of that degradation of the Slac2-a is essential for proper melanosome distribution in melanocytes. Our findings into the of the disassembly of the transport complex in melanosome for degradation is in mammalian melanocytes. We C. for and for and S. Kuroda for