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• W2020121457 abstract "Melanosomes are specialized organelles that undergo a dynamic process of transport along the melanocyte dendrite to the dendrite tip and transfer to keratinocytes. We hypothesized that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE), which are involved in membrane fusion, and rab3a, a GTP-binding protein involved in exocytosis in neuronal cells and in SNARE complex assembly, may play a part in melanosome transport and transfer. By reverse transcription–polymerase chain reaction we identified transcripts for rab3a, vesicle-associated membrane protein-2, synaptosome-associated proteins of 23 kDa and 25 kDa, and syntaxin-4 in murine melanocytic cells. We also showed that purified melanosome preparations contain rab3a and SNARE, including vesicle-associated membrane protein-2, syntaxin-4, synaptosome-associated proteins 23 kDa and 25 kDa, and the SNARE accessory protein, α-soluble N-ethylmaleimide-sensitive factor attachment protein. Ultraviolet radiation is a potent stimulus for melanosome transport and transfer. We show that ultraviolet radiation rapidly suppresses melanosome-associated rab3a expression and that this occurs at the protein and mRNA level. Finally, we show that vesicle-associated membrane protein-2 and synaptosome-associated protein 23 kDa coimmunoprecipitate from purified melanocytic cell membranes, suggesting that they form complexes. The presence of rab3a and SNARE on melanosomes, and of SNARE complexes in melanocytic cell membranes suggests that these proteins play a part in targeting melanosomes to the plasma membrane, to melanosome transfer to keratinocytes, or both. Melanosomes are specialized organelles that undergo a dynamic process of transport along the melanocyte dendrite to the dendrite tip and transfer to keratinocytes. We hypothesized that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE), which are involved in membrane fusion, and rab3a, a GTP-binding protein involved in exocytosis in neuronal cells and in SNARE complex assembly, may play a part in melanosome transport and transfer. By reverse transcription–polymerase chain reaction we identified transcripts for rab3a, vesicle-associated membrane protein-2, synaptosome-associated proteins of 23 kDa and 25 kDa, and syntaxin-4 in murine melanocytic cells. We also showed that purified melanosome preparations contain rab3a and SNARE, including vesicle-associated membrane protein-2, syntaxin-4, synaptosome-associated proteins 23 kDa and 25 kDa, and the SNARE accessory protein, α-soluble N-ethylmaleimide-sensitive factor attachment protein. Ultraviolet radiation is a potent stimulus for melanosome transport and transfer. We show that ultraviolet radiation rapidly suppresses melanosome-associated rab3a expression and that this occurs at the protein and mRNA level. Finally, we show that vesicle-associated membrane protein-2 and synaptosome-associated protein 23 kDa coimmunoprecipitate from purified melanocytic cell membranes, suggesting that they form complexes. The presence of rab3a and SNARE on melanosomes, and of SNARE complexes in melanocytic cell membranes suggests that these proteins play a part in targeting melanosomes to the plasma membrane, to melanosome transfer to keratinocytes, or both. soluble N-ethylmaleimide-sensitive factor attachment protein receptors vesicle-associated membrane protein Aunique function of the melanocyte–keratinocyte unit is the process of melanosome transfer. Melanocytes are pigment producing cells derived from the neural crest that reside in the basal layer of the epidermis, and extend multiple long dendritic processes towards epidermal keratinocytes. Melanosomes are melanocyte-specific membrane bound organelles that arise from coated vesicles from the Golgi apparatus and share a biosynthetic pathway with lysosomes (Maul and Brumbaugh, 1971Maul G.G. Brumbaugh J.A. On the possible function of coated vesicles in melanogenesis of the regenerating fowl feather.J Cell Biol. 1971; 48: 41-48Crossref PubMed Scopus (79) Google Scholar;Zhou et al., 1993Zhou B.K. Boissy R.E. Pifko-Hirst S. Moran D.J. Orlow S.J. Lysosome-associated membrane protein-1 (LAMP) is the melanocyte vesicular membrane glycoprotein band II.J Invest Dermatol. 1993; 100: 110-114Abstract Full Text PDF PubMed Google Scholar;Diment et al., 1995Diment S. Eidelman M. Rodriguez G.M. Orlow S.L. Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells.J Biol Chem. 1995; 270: 4213-4215Crossref PubMed Scopus (88) Google Scholar). Melanosome transport along the dendrite is mediated in part by microtubular proteins that translocate melanosomes to dendritic tips, and myosin V, which traps melanosomes at the actin-rich periphery of the dendrite (Provance et al., 1996Provance Jr, Dw Wei M. Ipe V. Mercer J.A. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution.Proc Natl Acad Sci USA. 1996; 93: 14554-14558https://doi.org/10.1073/pnas.93.25.14554Crossref PubMed Scopus (145) Google Scholar;Wei et al., 1997Wei Q. Wu X. Hammer III, J.A. The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape. Supporting a role for myosin V in melanosome transport.J Muscle Res Cell Motil. 1997; 18: 517-527Crossref PubMed Scopus (64) Google Scholar;Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.J Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar,Wu et al., 1998Wu X. Bowers B. Rao K. Wei Q. Hammer III, J.A. Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo.J Cell Biol. 1998; 143: 1899-1918Crossref PubMed Scopus (328) Google Scholar). The mechanisms underlying melanosome transfer are less well understood and may occur through a combination of processes, including phagocytosis of the melanocyte dendrite, exocytosis of the melanosome with uptake by keratinocytes, or both (Cohen and Szabo, 1967Cohen J. Szabo G. Study of pigment donation in vitro.Exp Cell Res. 1967; 50: 418-434Crossref Scopus (40) Google Scholar;Wolff, 1973Wolff K. Melanocyte–keratinocyte interactions in vivo: the fate of melanosomes.Yale J Biol Med. 1973; 46: 384-396PubMed Google Scholar;Yamamoto and Bhawan, 1994Yamamoto O. Bhawan J. Three modes of melanosome transfer in Caucasian facial skin: hypothesis based on an ultrastructural study.Pigment Cell Res. 1994; 7: 158-169Crossref PubMed Scopus (69) Google Scholar). Little is known about melanosome transfer at the molecular level, but membrane fusion between the melanosome and the plasma membrane of the melanocyte dendrite or the keratinocyte membrane during the process of transfer is likely to be involved. Rab3a and the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) family of proteins are involved in vesicle transport and docking (rab3a) and membrane fusion (SNARE). Because of the compelling role of rab3a and SNARE proteins in regulated exocytosis in neuronal cells and in other cell types, we investigated the expression and regulation of these proteins by ultraviolet (UV) irradiation in melanosomes. The rab family of GTP-binding proteins regulate vesicle transport as well as exocytosis (for review, seeJahn, 1999Jahn R. Membrane fusion and exocytosis.Annu Rev Biochem. 1999; 68: 863-911Crossref PubMed Scopus (988) Google Scholar) and consist of over 30 proteins that localize to the surface of organelles involved in vectorial transport. At least 17 rab cDNA have been identified in melanocytic cells by polymerase chain reaction (PCR) analysis (Chen et al., 1996Chen D. Guo J. Mike T. Tachibana M. Gahl W.A. Molecular cloning of two novel rab genes from human melanocytes.Gene. 1996; 174: 129-134https://doi.org/10.1016/0378-1119(96)00509-4Crossref PubMed Scopus (37) Google Scholar,Chen et al., 1997Chen D. Guo J. Gahl W.A.R.A.B. GTPases expressed in human melanoma cells.Biochim Biophys Acta. 1997; 1355: 1-6Crossref PubMed Scopus (10) Google Scholar) and Gomez et al 1Gomez PF, Luo D, Suzuki J, Otsu K, Ishikawa K, Jimbow K: A small molecular weight GTP-binding protein rab-7, is involved in the transport of TRP-1 from TGN to melanosomes. Pig Cell Res Suppl 5:35a 19961Gomez PF, Luo D, Suzuki J, Otsu K, Ishikawa K, Jimbow K: A small molecular weight GTP-binding protein rab-7, is involved in the transport of TRP-1 from TGN to melanosomes. Pig Cell Res Suppl 5:35a 1996 have implicated rab7 and rab5 in the transport of tyrosinase-related protein-1 (TRP-1) through the Golgi stack to the melanosome. Rab3a is primarily expressed in neurons and endocrine cells, and has significant homology to the yeast Sec4 protein, which is required for vesicular traffic from the trans-Golgi network to the plasma membrane (for review, seeHenry et al., 1996Henry J.-P. Johannes L. Dousseaut F. Poulain B. Darchen F. Role of Rab3a in neurotransmitter and hormone release: a discussion of recent data.Biochem Soc Trans. 1996; 24: 657-661Crossref PubMed Scopus (2) Google Scholar). Rab3a is located on the cytoplasmic surface of secretory granules or synaptic vesicles and like other rab proteins is isoprenylated on cysteine residues in the carboxy terminus by geranylgeranyl transferase II, which stabilizes membrane attachment (Desnoyers et al., 1996Desnoyers L. Anant J.S. Seabra M.C. Geranylgeranylation of Rab proteins.Biochem Soc Trans. 1996; 24: 699-703Crossref PubMed Scopus (46) Google Scholar). Rab3a has been shown to be critical for exocytosis of synaptic vesicles in neurons, secretion from pancreatic β-cells, granule secretion in mast cells and zymogen secretion from pancreatic acinar cells (Padfield et al., 1992Padfield P.J. Balch W.E. Jamieson J.D. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.Proc Natl Acad Sci USA. 1992; 89: 1656-1660Crossref PubMed Scopus (109) Google Scholar;Senyshyn et al., 1992Senyshyn J. Balch W.E. Holz R.W. Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells.FEBS Lett. 1992; 309: 41-46Abstract Full Text PDF PubMed Scopus (62) Google Scholar;Law et al., 1993Law G.J. Northrop A.J. Mason W.T. Rab3-peptide stimulates exocytosis from mast cells via a pertussis toxin-sensitive mechanism.FEBS Lett. 1993; 333: 56-60Abstract Full Text PDF PubMed Scopus (30) Google Scholar;Holz et al., 1994Holz R.W. Brondyk W.H. Senter R.A. Kuizon L. Macara I.G. Evidence for the involvement of Rab3A in Ca2+-dependent exocytosis from adrenal chromaffin cells.J Biol Chem. 1994; 269: 10229-10234Abstract Full Text PDF PubMed Google Scholar;Jena et al., 1994Jena B.P. Gumkowski F.D. Konieczko E.M. Fisher von Mollard G. Jahn R. Jamieson J.D. Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi-complex in pancreatic acinar cells during regulated exocytosis.J Cell Biol. 1994; 124: 43-53Crossref PubMed Scopus (86) Google Scholar;Regazzi et al., 1996Regazzi R. Ravazzola M. Iezzi M. et al.Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells.J Cell Sci. 1996; 109: 2265-2273PubMed Google Scholar). Rabphilin is a downstream effector molecule of rab3a and binds to the GTP-bound form of rab3a, as well as to α-actinin and β-adducin (Kato et al., 1996Kato M. Sasaki T. Ohya T. Nakanishi H. Nishioka H. Imamura M. Takai Y. Physical and functional interaction of rabphilin-3A with α-actinin.J Biol Chem. 1996; 271: 31775-31778Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar;Shirataki et al., 1993Shirataki H. Kaibuchi K. Sakoda T. et al.Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagminin.Mol Cell Biol. 1993; 13: 2061-2068Crossref PubMed Scopus (350) Google Scholar;Mivazaki et al., 1994Mivazaki M. Shirataki H. Kohno H. Kaibuchi K. Tsugita A. Takai Y. Identification as β-adducin of a protein interacting with Rabphilin-3a in the presence of calcium and phosphatidylserine.Biochem Biophys Res Commun. 1994; 205: 460-466https://doi.org/10.1006/bbrc.1994.2688Crossref PubMed Scopus (37) Google Scholar). It is highly concentrated on synaptic vesicles and functions with rab3a in Ca2+-dependent exocytosis. Rabphilin possesses sequence similarity with synaptotagmin, a Ca2+ sensor protein. Three alternative rab3a effector molecules have been identified termed NOC2, RIM, and rabin3 (Brondyk et al., 1995Brondyk W.H. McKiernan C.J. Fortner K.A. Stabila P. Holz R.W. Macara I.G. Interactive cloning of rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.Mol Cell Biol. 1995; 15: 1137-1143Crossref PubMed Google Scholar;Kotake et al., 1997Kotake K. Ozaki N. Mizuta M. Sekiya S. Inagaki N. Seino S. Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.J Biol Chem. 1997; 272: 29407-29410Crossref PubMed Scopus (79) Google Scholar;Wang et al., 1997Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion.Nature. 1997; 388: 593-598https://doi.org/10.1038/41580Crossref PubMed Scopus (512) Google Scholar;Kato et al., 1997Kato M. Sasaki T. Ohya T. et al.Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.J Biol Chem. 1997; 272: 29407-29410Crossref PubMed Scopus (75) Google Scholar). Whereas rab proteins determine vectorial transport and docking of vesicles among various cellular compartments, the SNARE family of proteins provides the apparatus necessary for the fusion of vesicle and target membranes (for review, seeLinial, 1997Linial M. SNARE proteins–-Why so many, why so few?.J Neurochem. 1997; 69: 1781-1792Crossref PubMed Scopus (80) Google Scholar). SNAREs were originally found in neurons and yeast, but homologs of the yeast and neuronal SNARE proteins have been identified in non-neuronal cells, including epithelial cells, platelets, adipose cells, pancreatic acinar cells, and other tissues (Cain et al., 1992Cain C.C. Trimble W.S. Lienhard G.E. Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.J Biol Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar;Wheeler et al., 1996Wheeler M.B. Sheu L. Ghai M. et al.Characterization of SNARE, protein expression in, β cell lines and pancreatic islets.Endocrinology. 1996; 137: 1341-1348Google Scholar;Ikebuchi et al., 1998Ikebuchi Y. Masumoto N. Matsuoka T. et al.SNAP-25 is essential for cortical granule exocytosis in mouse eggs.Am J Physiol. 1998; 274: 1496-1500Google Scholar;Quintanar et al., 1998Quintanar J.L. Salinas E. Reig J.A. Immunohistochemical demonstration of syntaxin and SNAP-25 in chromaffin cells of the frog adrenal gland.Gen Comp Endocrinol. 1998; 111: 119-122https://doi.org/10.1006/gcen.1998.7110Crossref PubMed Scopus (2) Google Scholar;Rea et al., 1998Rea S. Bartin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.J Biol Chem. 1998; 273: 18784-18792Crossref PubMed Scopus (92) Google Scholar;Flaumenhaft et al., 1999Flaumenhaft R. Croce K. Chen E. Furie B. Furie B.C. Proteins of the exocytotic core complex mediate platelet α-granule secretion.J Biol Chem. 1999; 274: 2492-2501Crossref PubMed Scopus (112) Google Scholar). SNAREs are mostly membrane-anchored proteins that share a common motif of approximately 60 amino acids. Synaptotagmins are a family of proteins that interact with the core SNARE complex and bind Ca2+. The binding of synaptotagmin to Ca2+ is believed to be crucial for the Ca2+ dependence of synaptic vesicle release, and results in the penetration of synaptotagmin into membranes and drives assembly of synaptotagmin into the SNARE complex (Davis et al., 1999Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lweis J.L. Chapman E.R. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Direct binding interactions between SNARE proteins in combination with accessory soluble proteins (such as synaptotagmin) result in the formation of a stable complex that acts as a receptor for α-SNAP [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein]. In the presence of NSF, a 20S particle is formed and following hydrolysis of adenosine triphosphate (ATP) by NSF, the SNARE complex is disrupted, which may facilitate membrane fusion (Sollner et al., 1993Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1528) Google Scholar;Pevsner et al., 1994Pevsner J. Hsu S.C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Specificity and regulation of a synaptic vesicle docking complex.Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). SNAP and NSF are universally shared cofactors for SNARE reactions and are required for all fusion events. The specificity of the membrane fusion reaction is provided by the SNARE proteins that serve as membrane receptors. On the basis of their localization on either vesicle or target membranes, SNAREs were originally classified as either vesicle SNAREs or target SNAREs. The prototype vesicle SNARE consists of the VAMP (vesicle-associated membrane protein)/synaptobrevin) family of proteins of which eight isoforms have been identified. VAMPs are small (18 kDa) proteins that anchor to the cytoplasmic surface of the vesicle by a carboxyl-terminal transmembrane domain. VAMP-1 is primarily present on synaptic vesicles, whereas VAMP-2 and VAMP-3 have been identified in fat and muscle tissues and in secretory cells (Gaisano et al., 1994Gaisano H.Y. Sheu L. Foskett J.K. Trimble W.S. Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion.J Biol Chem. 1994; 269: 17062-17066Abstract Full Text PDF PubMed Google Scholar;Volchuk et al., 1994Volchuk A. Mitsumoto Y. He L. Liu Z. Habernamm E. Trimble W. Klip A. Expression of vesicle-associated membrane protein 2 (VAMP-2) /synaptobrevin II and cellubrevin in rat skeletal muscle and in a muscle cell line.Biochem J. 1994; 320: 429-436Google Scholar,Volchuk et al., 1995Volchuk A. Sargeant R. Sumitani S. Lie Z. He L. Klip A. Cellubrevin is a resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in 3T3-L1 adipocytes.J Biol Chem. 1995; 270: 8233-8240Crossref PubMed Scopus (121) Google Scholar;Nielsen et al., 1995Nielsen S. Marples D. Birn H. Mohtashami M. Dalby N.O. Trimble W. Knepper M. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.J Clin Invest. 1995; 96: 1834-1844Crossref PubMed Scopus (135) Google Scholar;Timmers et al., 1996Timmers K.I. Clark A.E. Omatsu-Kanabe M. Whiteheart S.E. Bennet M.K. Holman G.D. Cushman S.W. Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein.Biochem J. 1996; 320: 429-436Crossref PubMed Scopus (54) Google Scholar;Wheeler et al., 1996Wheeler M.B. Sheu L. Ghai M. et al.Characterization of SNARE, protein expression in, β cell lines and pancreatic islets.Endocrinology. 1996; 137: 1341-1348Google Scholar). Recently a splice isoform of VAMP-1 has been described (VAMP-1b), which contains a mitochondrial targeting signal (Isenmann et al., 1998Isenmann S. Khew-Goodall Y. Gamble J. Vadas M. Wattenberg B.W. A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.Mol Biol Cell. 1998; 9: 1649-1690Crossref PubMed Scopus (111) Google Scholar). Using PCR analysis, VAMP-1a is exclusively present in brain tissue, whereas VAMP-1b is present in endothelial cell lines and other non-neuroneal cell lines (Isenmann et al., 1998Isenmann S. Khew-Goodall Y. Gamble J. Vadas M. Wattenberg B.W. A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.Mol Biol Cell. 1998; 9: 1649-1690Crossref PubMed Scopus (111) Google Scholar). Syntaxins are target SNAREs of which 17 isoforms have been identified to date. Syntaxins are approximately 35 kDa membrane proteins that localize to the plasma membrane and in the case of syntaxin-1 and syntaxin-4, have been shown to bind the VAMP family of proteins. SNAP-25 (synaptosome-associated protein of 25 kDa) is a plasma membrane protein identified on developing and mature neurons that associates with syntaxin and VAMP, as well as synaptotagmin, to regulate exocytosis in neuroneal cells, as well as axonal extension in developing neurons (Osen-Sand et al., 1993Osen-Sand A. Catsicas M. Staple J.K. et al.Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo.Nature. 1993; 364: 445-448Crossref PubMed Scopus (375) Google Scholar). Two SNAP-25 isoforms (a and b) have been identified in chicken, human, and mouse tissue (Bark, 1993Bark I.C. Structure of the chicken gene for SNAP-25 reveals duplicated exon encoding distinct isoforms of the protein.J Mol Biol. 1993; 233: 67-76https://doi.org/10.1006/jmbi.1993.1485Crossref PubMed Scopus (102) Google Scholar;Bark and Wilson, 1994Bark I.C. Wilson M.C. Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25.Gene. 1994; 139: 291-292Crossref PubMed Scopus (131) Google Scholar). These two isoforms differ by only nine amino acids and are generated by alternative splicing of exons 5a and 5b. SNAP-23 is 59% identical and 72% similar to SNAP-25 at the amino acid level (Ravichandran et al., 1996Ravichandran V. Chawla A. Roche P.A. Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.J Biol Chem. 1996; 271: 13300-13303Crossref PubMed Scopus (281) Google Scholar) and the regions of highest homology of SNAP-23 to SNAP-25 reside in the amino- and carboxyl-terminal thirds of the SNAP-23 protein. SNAP-23 has been reported to replace SNAP-25 in the process of insulin secretion when it is overexpressed (Sadoul et al., 1997Sadoul K. Berger A. Niemann H. et al.SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion.J Biol Chem. 1997; 272: 33023-33027Crossref PubMed Scopus (88) Google Scholar), to mediate mast cell secretion, and to play a part in vesicle transport in non-neuronal cell types (Guo et al., 1998Guo Z. Turner C. Castle D. Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells.Cell. 1998; 94: 537-548Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Whereas rab proteins are not components of the SNARE complex, they, or their effector molecules, participate in SNARE assembly, membrane fusion, and specificity of vesicle and target membrane fusion (Lian et al., 1994Lian J.P. Stone S. Jiang Y. Lyons P. Ferro-Novick S. Ypt 1p implicated in V-SNARE activation.Nature. 1994; 372: 698-701Crossref PubMed Scopus (160) Google Scholar;Sogaard et al., 1994Sogaard M. Tani K. Ye R.R. et al.A rab protein is required for the assembly of SNARE, complexes in the docking of transport vesicles.Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (433) Google Scholar;Lupashin and Waters, 1997Lupashin V.V. Waters M.G. t-SNARE activation through transient interaction with a rab-like guanosine triphosphatase.Science. 1997; 276: 1255-1258https://doi.org/10.1126/science.276.5316.1255Crossref PubMed Scopus (182) Google Scholar;McBride et al., 1999McBride H.M. Rybin V. Murphy C. Giner A. Teasdale R. Zerial M. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.Cell. 1999; 98: 337-386Abstract Full Text Full Text PDF Scopus (384) Google Scholar). Rab3a in particular has been shown to regulate the formation and stability of the SNARE complex (Johannes et al., 1996Johannes L. Doussau F. Clabecq A. Henry J. Darchen F. Poulain B. Evidence for a functional link between Rab3 and the SNARE complex.J Cell Sci. 1996; 109: 2875-2884Crossref PubMed Google Scholar). Because melanosome transfer to keratinocytes may be conceptualized as an exocytotic process and because melanocytes are derived from the neural crest, it may be expected that SNARE proteins would be expressed in melanocytic cells and would play a part in melanosome dynamics. Because rab3a has been implicated in exocytosis in neuroendocrine cells as well as other cell types and has been implicated in SNARE assembly, we determined its expression and regulation by UV radiation, a potent stimulus for melanosome transfer. Finally, we studied VAMP-2 and SNAP-23 association in irradiated melanocytic cells. Our results show that SNARE proteins and rab3a are present in enriched melanosome fractions and that UV radiation regulates rab3a expression. We also show that the vesicle and target SNARE proteins VAMP-2 and SNAP-23 are associated in UV-radiated melanocytic cells. These findings suggest that rab3a and SNARE proteins play a part in melanosome dynamics. Polyclonal antibodies to rab3a and VAMP-2, and monoclonal antibodies to α-SNAP were obtained from StressGen Biotechnologies (Victoria, Canada); polyclonal antibodies to SNAP-23 were obtained from Synaptic Systems (Gottingen, Germany); monoclonal antibodies to SNAP-25 were obtained from Chemicon International (Temecula, CA); and monoclonal antibodies to syntaxin-4 and rabphilin-3a were obtained form Transduction Laboratories (Lexington, KY). Positive controls for western blotting for rab3a, VAMP-2, α-SNAP, and rabphilin-3a consisted of mouse brain tissue extracts (StressGen Biotechnologies); positive controls for western blotting for syntaxin-4 and SNAP-23 consisted of cultured human endothelial cell extracts (Transduction Laboratories). Human endothelial cells were derived from aortic endothelium and were cultured in Dulbecco's minimal essential media (Gibco BRL, Gaithersburg, MD) and 10% fetal bovine serum (Gibco BRL). For dual localization with the melanosomal transmembrane protein TRP-1 polyclonal antibodies to TRP-1 (clone αPEP1) were used and were a generous gift of Dr. Vincent Hearing (National Institutes of Health, Bethesda MD) and have been described previously (Jimenez et al., 1988Jimenez M. Kameyama K. Maloy W.L. Tomita Y. Hearing V.J. Mammalian tyrosinase: biosynthesis, processing and modulation by melanocyte stimulating homone.Proc Natl Acad Sci USA. 1988; 85: 3830-3834Crossref PubMed Scopus (121) Google Scholar). α-PEP1 was generated against a fragment of the carboxy terminal of murine TRP-1 and does not cross-react with human TRP-1 (Jimenez et al., 1988Jimenez M. Kameyama K. Maloy W.L. Tomita Y. Hearing V.J. Mammalian tyrosinase: biosynthesis, processing and modulation by melanocyte stimulating homone.Proc Natl Acad Sci USA. 1988; 85: 3830-3834Crossref PubMed Scopus (121) Google Scholar). Monoclonal antibodies to the α1 subunit of the Na+/K+ ATPase were obtained from Affinity Biogreagents (Golden, CO); positive controls for Na+/K+ ATPase consisted of canine skeletal muscle microsomes and supernatant and were purchased from Affinity Bioreagents. Goat anti-mouse IgG and anti-rabbit horseradish peroxidase conjugated antibodies were obtained from Pierce (Rockford, IL); Texas red-conjugated and fluorescein isothiocyanate-conjugated secondary antibodies were purchased from Molecular Probes (Eugene OR); normal goat serum and normal rabbit serum and all chemicals were obtained from Sigma (St Louis, MO). B16F10 murine melanoma cells were obtained from American Type Culture Collection (ATCC) and were maintained in Dulbecco's minimal essential media + 10% fetal bovine serum. B10BR murine immortalized melanocytes were a generous gift of Dr. Ruth Halaban (Yale University School of Medicine) and were maintained in Ham's F-10 media (Gibco BRL) supplemented with 10% horse serum, 50 ng per ml TPA (Sigma), 2% fetal bovine serum, and 50 μg per ml gentamycin (Gibco BRL). HaCat cells (human immortalized keratinocytes) were obtained from ATCC and were maintained in Dulbecco's minimal essential media + 10% fetal bovine serum. Human keratinocytes were cultured from newborn foreskins as previously described (Scott and Haake, 1991Scott G.A. Haake A.R. Keratinocytes regulate melanocyte number in human fetal and neonatal skin equivalents.J Invest Dermatol. 1991; 97: 776-781Abstract Full Text PDF PubMed Google Scholar) in keratinocyte growth media (Clonetics, San Diego, CA). The source of UV irradiation was a 1000 W Oriel Xenon Arc Solar Simulator with UVC WG320 and visible cut-off filter UG11, beam turner, and cooling fan. This radiation source emits electromagnetic radiation from 295 nm (UVB) to 400 nm (UVA) and produces 3.75 × 10-3 W per cm2. An IL 1700 radiometer and SED400 sensor probe purchased from International Light (Newburyport, MA) monitored the output. The desired dose of UV radiation (1.1 J per cm2) was obtained by irradiating the cells for 5 min. During UV irradiation, medium was replaced with phosphate-buffered saline containing Ca2+ and Mg2+. The phosphate-buffered saline was then replaced with growth medium. Cells were collected by trypsinization and homogenized in hypotonic homogenization buffer (10 mM Tris–HCl, 10 mM KCl. 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 0.05 mM MgCl2)" @default.
• W2020121457 created "2016-06-24" @default.
• W2020121457 creator A5001559303 @default.
• W2020121457 creator A5085366413 @default.
• W2020121457 date "2001-02-01" @default.
• W2020121457 modified "2023-10-18" @default.
• W2020121457 title "Rab3a and SNARE Proteins: Potential Regulators of Melanosome Movement" @default.
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