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• W2016201659 startingPage "43096" @default.
• W2016201659 abstract "Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A·Slac2-a·myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432–12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B bothin vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400–590) and Slac2-c (amino acids 670–856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery. Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A·Slac2-a·myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432–12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B bothin vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400–590) and Slac2-c (amino acids 670–856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery. synaptotagmin-like protein brain glutathioneS-transferase globular tail horseradish peroxidase melanocyte 5′-rapid amplification of cDNA ends reverse transcriptase Slp homology domain expressed sequence tag green fluorescent protein The Rab family, one of the small GTP-binding protein subfamilies (1Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (374) Google Scholar), is thought to control intracellular membrane trafficking in eukaryotic cells (reviewed in Refs. 2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). More than 60 distinct Rab proteins have been identified in humans (5Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (521) Google Scholar, 6Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2001; 313: 889-901Crossref PubMed Scopus (611) Google Scholar), and they seem to regulate various steps of membrane trafficking (e.g. vesicle formation, docking, tethering, and fusion) (2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Although Rab proteins are generally believed to act with specific effector molecule(s) that preferentially bind the GTP-bound activated form of Rab, only a limited number of effector molecules have been identified to date (2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), and the exact roles of most of the Rab proteins remain to be elucidated. Recent studies have suggested that several Rabs (i.e. Rab6, Rab11, and Rab25) are involved in the movement of transport vesicles from their site of formation to their site of fusion, because these Rabs have been found to interact directly with specific microtubule- or actin-based motor proteins (see Refs. 7Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (411) Google Scholar, 8Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (342) Google Scholar, 9Schott D., Ho, J. Pruyne D. Bretscher A. J. Cell Biol. 1999; 147: 791-808Crossref PubMed Scopus (199) Google Scholar, and reviewed in Ref. 10Hammer III., J.A. Wu X.S. Curr. Opin. Cell Biol. 2002; 14: 69-75Crossref PubMed Scopus (124) Google Scholar). For instance, Rab6 interacts with the C-terminal domain of Rabkinesin-6 (7Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (411) Google Scholar), whereas Rab11 and Rab25 interact with the C-terminal domain of the myosin Vb tail (8Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (342) Google Scholar). Very recently, another type of Rab-motor protein interaction has been discovered in melanosome transport (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 12Wu 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 (380) Google Scholar, 13Hume 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 (132) Google Scholar, 14Provance Jr., D.W. James T.L. Mercer J.A. Traffic. 2002; 3: 124-132Crossref PubMed Scopus (111) Google Scholar) and plasma membrane recycling systems (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar); myosin Va indirectly recognizes Rab27A on melanosomes via Slac2-a (synaptotagmin-like protein (Slp)1 homologuelacking C2 domains-a) (also called melanophilin), a linker protein that interacts specifically and directly with the GTP-bound form of Rab27A and myosin Va (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 12Wu 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 (380) Google Scholar, 16Kuroda 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,17Fukuda M. J. Biol. Chem. 2002; 277: 40118-40124Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas myosin Vb interacts with Rab11 (and Rab25), as well as Rab11-FIP2 (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Although five members of the Rab11-FIP family have been described to date (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 18Prekeris R. Davies J.M. Scheller R.H. J. Biol. Chem. 2001; 276: 38966-38970Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 19Lindsay A.J. Hendrick A.G. Cantalupo G. Senic-Matuglia F. Goud B. Bucci C. McCaffrey M.W. J. Biol. Chem. 2002; 277: 12190-12199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), no information is available for the existence of a Slac2-a homologue (or Slac2 family). Because Rab27A and myosin Va are known to be expressed in tissues other than melanocytes, we hypothesized that other linker protein(s) must exist in the body. In this paper, we report on a novel Slac2-a homologue (named Slac2-c) that interacts specifically with Rab27A/B and myosin Va/VIIa by means of EST database searches and biochemical experiments. We also discovered that the conserved most C-terminal region of Slac2 functions as a novel actin-binding site. Based on our findings, we discuss the role of Slac2 in Rab27A-dependent membrane trafficking. cDNAs encoding the N-terminal region of Slac2-c were amplified from Marathon-Ready adult mouse brain cDNA (Clontech Laboratories, Inc.; Palo Alto, CA) by 5′-rapid amplification of cDNA ends (RACE) as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). First 5′-RACE reactions were carried out using the adapter primer 1 (5′-CCATCCTAATACGACTCACTATAGGGC-3′) and Slac2-c-stop primer (5′-TTAGTACATCACAGCTGACT-3′; termination codon is shown in boldface letters) designed on the basis of rat and mouse EST sequences (GenBankTM accession numbers BF287121 and BG869374). Second RACE reactions were carried out using the internal adapter primer 2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′) and Slac2-c C1 primer (5′-GTGCTGGACCGGGAATTCTG-3′). The purified PCR products were inserted directly into the pGEM-T Easy vector (Promega; Madison, WI), and both strands were sequenced completely with a Hitachi SQ-5500 DNA sequencer as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). cDNAs encoding the open reading frame of the mouse Slac2-c were amplified by reverse transcriptase (RT)-PCR from Marathon-Ready adult mouse brain cDNA with Slac2-c Met (5′-GGATCCATGGGGAGGAAGCTGGACCT-3′;BamHI site is underlined) and Slac2-c-stop primers. The purified PCR products were subcloned into the pGEM-T Easy vector (named pGEM-T-Slac2-c) and verified by DNA sequencing. Addition of the T7 tag to the N terminus of Slac2-c and construction of the mammalian cell expression vector (named pEF-T7-Slac2-c) were performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 22Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). The human Slac2-c cDNA was determined by database searching (standard BLAST search) using the mouse Slac2-c as a query. cDNA encoding a tail domain of mouse myosin VIIa (23Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar) or MC-type myosin Va (− exon B, + exon D, and + exon F) (24Seperack 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, 25Huang 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) was amplified from Marathon-Ready adult brain cDNA or spleen cDNA from mouse MTC Panel I (Clontech Laboratories, Inc.), respectively, by RT-PCR as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda 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, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The following oligonucleotides were used for amplification of the mouse myosin VIIa cDNA: 5′-CGGATCCCGGGTTGAGTACCAGCGGCG-3′ (myosin VIIa-N1 primer, sense), 5′-GATGTGGTCAATTATGCCCG-3′ (myosin VIIa- N2 primer, sense), 5′-CTCAAGTACATGGGCGACTA-3′ (myosin VIIa-N3 primer, sense), 5′-GCCTGAGAATTTGTAAGCTT-3′ (myosin VIIa-C1 primer, antisense), 5′-CTCAAAGATCTGGTCAGTGA-3′ (myosin VIIa-C2 primer, antisense), and 5′-TCACCTCCCGCTCCTGGAGT-3′ (myosin VIIa-stop primer, antisense). The purified PCR products were inserted directly into the pGEM-T Easy vector and were sequenced completely. We identified one amino acid difference (L1156F) compared with the reported myosin VIIa sequences (GenBankTM accession number NM_008663) (23Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar), and it is unlikely to be a PCR-induced error, because we found the same difference in at least two independent clones. Addition of the FLAG tag to the N terminus of myosin VIIa tail or MC-type myosin Va tail and construction of the expression vector (pEF-FLAG-myosin VIIa-tail and pEF-FLAG-MC-myosin Va-tail) were performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 22Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). pEF-T7-GST-FLAG-MC-myosin Va-F-GT (MC-specific exon F + globular tail) and pEF-FLAG-MC-myosin Va-F-GT were constructed by PCR essentially as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Brain (BR)-type myosin Va cDNA (+ exon B, − exon D, and − exon F) (pEF-FLAG-BR-myosin Va) was prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Deletion mutants of Slac2-a (pEF-T7-Slac2-a-Δ146/481, -Δ146/321, -Δ400, and -Δ240) and of Slac2-c (pEF-T7-Slac2-c-SHD, -ΔSHD, -Δ146/494, -495/856, and pEF-T7-GST-Slac2-c-ΔSHD) were constructed essentially by conventional PCR as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) using the following oligonucleotides with restriction enzyme sites (underlined) or stop codons (in bold): 5′-CGGATCCGAGGGCCTAGAGGAGACTGG-3′ (Slac2-a-Δ240 primer; sense), 5′-CGGATCCTCATCAGAAGATGAGACCAA-3′ (Slac2-a-Δ400 primer; sense), 5′-GCACTAGT CAAGCTGCGATCCTGGACTGGA-3′ (Slac2-a-Δ481 primer; antisense), 5′-GCACTAGT CAACCCTGGATACTGTCTTCAT-3′ (Slac2-a-Δ321 primer; antisense), 5′-GCACTAGT CAGGGCTCCAGAGAGGTGGTGT-3′ (Slac2-a-Δ241 primer; antisense), 5′-CGGATCCCGTCTGGAGAGCGGTGCCTG-3′ (Slac2-c-ΔSHD primer; sense), 5′-CGGATCCTTCAACCCTCAGGCAGCCGG-3′ (Slac2-c-Δ494 primer; sense), 5′-TCAGTGTTTTCTGTACAGGTTCT-3′ (Slac2-c-Δ146 primer, antisense), and 5′-TCAATTGACGTCCAGTTCTCCCT-3′ (Slac2-c-Δ495 primer, antisense). pEGFP-C1-Slac2-a, -Slac2-a-SHD, and -Slac2-a-Δ400 (Clontech Laboratories, Inc.) were constructed similarly by PCR. Other expression constructs (pEF-FLAG-Rab1, -Rab2, -Rab3A, -Rab4A, -Rab5A, -Rab6A, -Rab7, -Rab8, -Rab9, -Rab10, -Rab11A, -Rab17, -Rab18, -Rab20, -Rab22, -Rab23, -Rab25, -Rab27A, -Rab27B, -Rab28, -Rab34, -Rab37, and pEF-T7-GST-FLAG-myosin Va-GT) were prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda 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). Mutant Rab27A plasmids carrying a Thr to Asn substitution at amino acid position 23 (T23N) (dominant negative form) or a Q78L substitution (dominant active form) were produced by PCR using the following mutagenic oligonucleotides and SP6 primer: 5′-CCTTGGGAGACTCTGGGGTAGGGAAGAACAGT-3′ (T23N primer) or 5′-CTGCAGTTATGGGACACGGCGGGGCTGGAG-3′ (Q78L primer). pGEM-T-Rab27A was used as a template (16Kuroda 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). The mutant Rab27A fragments were subcloned into the pEF-FLAG-tag vector (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) using appropriate restriction enzyme sites (underlined above). Mouse first-strand cDNAs prepared from various tissues and developmental stages were obtained from Clontech Laboratories, Inc. (mouse MTC Panel I) (28Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar, 29Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar). PCRs were carried out in the presence of Perfect Match PCR enhancer (Stratagene; La Jolla, CA) for 30 cycles (for G3PDH), 35 cycles (for Slac2-a), or 40 cycles (for Slac2-c), each consisting of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. Slac2-a-Δ146 primer (16Kuroda 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 Slac2-a-Δ321 primer and Slac2-c-N1 primer (5′-AGAGACTGACATCAGCAACG-3′) and Slac2-c-stop primer were used for amplification. The PCR products were analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. The authenticity of the products was verified by subcloning into a pGEM-T Easy vector and DNA sequencing as described above. GST-Slac2-a-Δ400 and T7-GST-Slac2-a-Δ146 were prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). G-actin and F-actin (Sigma) were prepared as described by Perelroizen et al. (30Perelroizen I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar) and were immobilized with anti-actin mouse monoclonal antibody (C-2)-conjugated agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Approximately 1 μg of GST-Slac2-a-Δ400 (or GST alone) were incubated with the G-actin (or F-actin) beads in G-buffer (5 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 0.2 mm ATP, and 0.1 mm CaCl2) or F-buffer (5 mmTris-HCl, pH 7.5, 1 mm dithiothreitol, 0.2 mmATP, 0.1 mm CaCl2, 2 mmMgCl2, and 0.1 m KCl) at 4 °C for 1 h and then washed five times with 1 ml of the binding buffer without actin. Proteins that bound the beads were analyzed by 10% SDS-PAGE and then immunoblotted with anti-actin goat polyclonal antibody (1/200 dilution) and horseradish peroxidase (HRP)-conjugated anti-GST antibody (1/2000 dilution; Santa Cruz Biotechnology, Inc.) as described previously (16Kuroda 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, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). New Zealand White rabbits were immunized with GST-Slac2-a-SHD (amino acids 1–153) (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda 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 anti-Slac2-a-SHD antibody was affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories, Hercules, CA) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Specificity of the antibody was checked by immunoblotting with recombinant T7-tagged Slac2-a and Slac2-c expressed in COS-7 cells (32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar, 33Fukuda M. Kowalchyk J.A. Zhang X. Martin T.F.J. Mikoshiba K. J. Biol. Chem. 2002; 277: 4601-4604Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 34Zhang X. Kim-Miller M.J. Fukuda M. Kowalchyk J.A. Martin T.F.J. Neuron. 2002; 34: 599-611Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Melanoma cells (B16-F1; 10-cm dish) were homogenized in a buffer containing 1 ml of the F-buffer and protease inhibitors, and proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removal of insoluble material by centrifugation, the supernatant was incubated with either anti-Slac2-a-SHD IgG (10 μg/ml) or control rabbit IgG for 1 h at 4 °C followed by incubation with protein A-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. After washing the beads five times with the F-buffer containing 0.2% Triton X-100 and protease inhibitors, the immunoprecipitates were subjected to 10% SDS-PAGE followed by immunoblotting with anti-actin (1/200 dilution) and anti-myosin Va goat antibodies (1/100 dilution; Santa Cruz Biotechnology, Inc.) as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). PC12 cells were cultured on glass-bottom dishes (35-mm dish; MatTek Corp., Ashland, MA) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A 4-μg sample of pEF-T7-Slac2-c-495/856, pEGFP-C1-Slac2-a-SHD, or -Slac2-a-Δ400 was transfected into PC12 cells by using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Exposure to nerve growth factor (Merck KGaA, Darmstadt, Germany) was performed as described previously (32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar). Melanoma cell line B16-F1 was cultured as described previously (16Kuroda 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). pEGFP-C1-Slac2-a was transfected into melanoma cells by using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. Three days after transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 2 min, incubated for 1 h at room temperature with anti-T7 tag (1/4000 dilution; Novagen, Madison, WI) or anti-Rab27A mouse monoclonal antibody (1/250 dilution; Transduction Laboratories; Lexinton, KY), and visualized by a second antibody (1/10,000 dilution; anti-mouse Alexa 488 antibodies; Molecular Probes, Inc.; Eugene, OR) or Texas Red-conjugated phalloidin (1/200 or 1/500 dilution; Molecular Probes, Inc.) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar). The cells were then examined with a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). Images were pseudo-colored and superimposed with Adobe Photoshop software (version 5.0). Cotransfection of pEF-T7-Slac2-c and pEF-FLAG-Rabs (or -FLAG-myosin Va) into COS-7 cells (7.5 × 105 cells, the day before transfection/10-cm dish) was performed as described previously (26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Proteins were solubilized with a buffer containing 1% Triton X-100, 250 mm NaCl, 1 mm MgCl2, 50 mm HEPES-KOH, pH 7.2, 0.1 mm phenylmethylsulfonyl fluoride, 10 μmleupeptin, and 10 μm pepstatin A at 4 °C for 1 h. T7-Slac2-c was immunoprecipitated with anti-T7 tag antibody-conjugated agarose (Novagen) as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 27Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Direct interaction of myosin Va-GT with Slac2-c-ΔSHD and in vitro formation of the tripartite protein complex (Slac2-c·Rab27A·myosin Va-F-GT) were also performed as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). SDS-PAGE and immunoblotting analyses with HRP-conjugated anti-FLAG tag (Sigma) and anti-T7 tag antibodies (Novagen) were also performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The blots and gels shown are representative of at least two or three independent experiments. Slac2 was identified originally as a protein that contains an N-terminal SHD without tandem C2 domains (29Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 35McAdara Berkowitz J.K. Catz S.D. Johnson J.L. Ruedi J.M. Thon V. Babior B.M. J. Biol. Chem. 2001; 276: 18855-18862Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 37Ishikawa K. Nagase T. Suyama M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 169-176Crossref PubMed Scopus (170) Google Scholar). Two forms of Slac2 (Slac2-a and Slac2-b/KIAA0624) have been reported in humans, but they do not show any significant homology except for the SHD (16Kuroda 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). Recently, Slac2-a was identified as melanophilin, and a mutation in themlph gene was discovered to cause defects in melanosome transport in leaden mice exhibiting a lighter coat color (i.e. accumulation of melanosomes in the perinuclear region) (38Matesic 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 (201) Google Scholar). Slac2-a/melanophilin has been shown to function as a linker protein that bridges between Rab27A on melanosomes (39Menasche 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 (739) Google Scholar, 40Bahadoran P. Aberdam E. Mantoux F. Busca R. Bille K. Yalman N. de Saint-Basile G. Casaroli-Marano R. Ortonne J.P. Ballotti R. J. Cell Biol. 2001; 152: 843-850Crossref PubMed Scopus (174) Google Scholar, 41Wilson 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 (339) Google Scholar, 42Hume A.N. Collinson L.M. Rapak A. Gomes A.Q. Hopkins C.R. Seabra M.C. J. Cell Biol. 2001; 152: 795-808Crossref PubMed Scopus (274) Google Scholar) and myosin Va (43Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (243) Google Scholar, 44Sellers J.R. Biochim. Biophys. Acta. 2000; 1496: 3-2" @default.
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