Structure-Function Analysis of VPS9-Ankyrin-repeat Protein (Varp) in the Trafficking of Tyrosinase-related Protein 1 in Melanocytes
2010; Elsevier BV; Volume: 286; Issue: 9 Linguagem: Inglês
10.1074/jbc.m110.191205
ISSN1083-351X
AutoresKanako Tamura, Norihiko Ohbayashi, Koutaro Ishibashi, Mitsunori Fukuda,
Tópico(s)RNA regulation and disease
ResumoBecause Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking. Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking. IntroductionPigmentation of mammalian hair and skin requires proper formation and transport of melanosomes, one of the lysosome-related organelles that specifically synthesize and store melanin pigments, in melanocytes (reviewed in Refs. 1Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (348) Google Scholar, 2Raposo G. Marks M.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 786-797Crossref PubMed Scopus (364) Google Scholar). Defects in the formation and/or transport of melanosomes often cause pigmentary disorders, e.g. albinism and pigmentary dilution, in mammals (reviewed in Ref. 3Tomita Y. Suzuki T. Am. J. Med. Genet. 2004; 131C: 75-81Crossref PubMed Scopus (120) Google Scholar). The formation and transport of melanosomes involve a variety of intracellular membrane trafficking events, and several distinct Rab-type small GTPases, which are conserved membrane trafficking proteins in all eukaryotic cells (reviewed in Refs. 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 5Fukuda M. Cell. Mol. Life Sci. 2008; 65: 2801-2813Crossref PubMed Scopus (301) Google Scholar, 6Stenmark H. Nat. Rev. Mol. Cell Biol. 2009; 10: 513-525Crossref PubMed Scopus (2221) Google Scholar), have been shown to regulate the maturation and transport of melanosomes in mammalian epidermal melanocytes.The best characterized Rab isoform that is abundant on mature melanosomes in melanocytes is Rab27A (7Hume 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 (272) Google Scholar, 8Bahadoran P. Aberdam E. Mantoux F. Buscà 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 (171) Google Scholar, 9Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer 3rd, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). Rab27A regulates actin-based melanosome transport through interaction with its specific effector, Slac2-a/melanophilin (10Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 11Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer 3rd, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (378) Google Scholar, 12Strom 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 (252) Google Scholar), and melanosome anchoring to the plasma membrane through interaction with another effector, Slp2-a (13Kuroda T.S. Fukuda M. Nat. Cell Biol. 2004; 6: 1195-1203Crossref PubMed Scopus (131) Google Scholar). As a result, Rab27A deficiency causes human type 2 Griscelli syndrome, which is characterized by silvery hair (i.e. partial albinism) (reviewed in Ref. 14Van Gele M. Dynoodt P. Lambert J. Pigment Cell Melanoma Res. 2009; 22: 268-282Crossref PubMed Scopus (116) Google Scholar and references therein). Two other Rab isoforms, Rab32 and Rab38, regulate an early step in melanogenesis, i.e. the transport of melanogenic enzymes to melanosomes (15Loftus S.K. Larson D.M. Baxter L.L. Antonellis A. Chen Y. Wu X. Jiang Y. Bittner M. Hammer 3rd, J.A. Pavan W.J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 4471-4476Crossref PubMed Scopus (138) Google Scholar, 16Park M. Serpinskaya A.S. Papalopulu N. Gelfand V.I. Curr. Biol. 2007; 17: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Wasmeier C. Romao M. Plowright L. Bennett D.C. Raposo G. Seabra M.C. J. Cell Biol. 2006; 175: 271-281Crossref PubMed Scopus (209) Google Scholar). Actually, dysfunction of Rab38 causes the diluted coat color of chocolate mice, presumably because of impairment of the targeting of tyrosinase-related protein 1 (Tyrp1) to melanosomes (15Loftus S.K. Larson D.M. Baxter L.L. Antonellis A. Chen Y. Wu X. Jiang Y. Bittner M. Hammer 3rd, J.A. Pavan W.J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 4471-4476Crossref PubMed Scopus (138) Google Scholar). The discovery of the Rab32/38-specific binding protein Varp 4The abbreviations used are: Varp, VPS9-ankyrin-repeat protein; ANK, ankyrin; EGFP, enhanced green fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate. (VPS9-ankyrin-repeat protein; official symbol in the National Center for Biotechnology Information is Ankrd27) has recently been reported (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar, 19Wang F. Zhang H. Zhang X. Wang Y. Ren F. Zhang X. Zhai Y. Chang Z. Biochem. Biophys. Res. Commun. 2008; 372: 162-167Crossref PubMed Scopus (30) Google Scholar), and as its name indicates, Varp protein contains an N-terminal VPS9 (vacuolar protein sorting 9) domain and C-terminal tandem ankyrin repeat domains (named ANKR1 and ANKR2). The Varp VPS9 domain possesses Rab21-GEF (guanine nucleotide exchange factor) activity (20Zhang X. He X. Fu X.Y. Chang Z. J. Cell Sci. 2006; 119: 1053-1062Crossref PubMed Scopus (77) Google Scholar), and the ANKR1 domain functions as a specific GTP-Rab32/38-binding site (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar, 19Wang F. Zhang H. Zhang X. Wang Y. Ren F. Zhang X. Zhai Y. Chang Z. Biochem. Biophys. Res. Commun. 2008; 372: 162-167Crossref PubMed Scopus (30) Google Scholar). Even more recently, VAMP7/TI-VAMP (vesicle-associated membrane protein) has been shown to interact with the region between the ANKR1 domain and the ANKR2 domain and to regulate the neurite outgrowth of hippocampal neurons (21Burgo A. Sotirakis E. Simmler M.C. Verraes A. Chamot C. Simpson J.C. Lanzetti L. Proux-Gillardeaux V. Galli T. EMBO Rep. 2009; 10: 1117-1124Crossref PubMed Scopus (73) Google Scholar), but whether the Varp·VAMP7 complex is involved in melanogenesis has never been demonstrated. Because knockdown of Varp or overexpression of the ANKR1 domain, i.e. Rab32/38-binding site, in cultured melanocytes causes the disappearance of Tyrp1 signals from peripheral melanosomes (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar), Varp is likely to be involved in trafficking of Tyrp1 in concert with Rab32/38. However, the functional interaction between Rab32/38 and Varp (or VAMP7 and Varp) and the involvement of Rab21-GEF activity in Tyrp1 trafficking are not fully understood, and the structural basis of the molecular recognition of Rab32/38 by the Varp ANKR1 domain, including the residue(s) that are critical for Rab32/38 (or ANKR1) recognition, has never been elucidated.In this study, we identified critical residues for the Rab32/38-Varp interaction in Rab32/38 (or Varp) by performing Ala-based site-directed mutagenesis. A switch II mutant of Rab38, which carries a Val-to-Ala mutation at amino acid position 78 (named Rab38(V78A)), completely lost its Varp binding activity without any change in its intrinsic GTPase activity or subcellular localization. We showed that whereas wild-type Rab38 is able to restore the peripheral melanosomal localization of Tyrp1 in Rab32/38-deficient melanocytes, the Rab38(V78A) mutant is incapable of rescuing Tyrp1-trafficking deficiency and that ANKR1 point mutants, Varp(Q509A) and Varp(Y550A), are also unable to rescue Tyrp1 deficiency in Varp-deficient melanocytes. We then investigated two Varp mutants carrying a point mutation in the VPS9 domain and a Varp deletion mutant lacking the VAMP7-binding site (named ΔVID) and showed that VAMP7 binding to Varp is required for Tyrp1 trafficking, but that Rab21-GEF activity is not. We discuss the structure-function relationships of Varp in Tyrp1 trafficking to melanosomes in melanocytes on the basis of these findings.DISCUSSIONWe have previously shown that the Rab32/38-specific binding protein Varp is expressed in cultured melanocytes and that its knockdown by a specific shRNA caused a dramatic reduction in Tyrp1 signals (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar), suggesting that a Varp·Rab32/38 complex regulates the transport of Tyrp1 to melanosomes, but whether interaction between Varp and Rab32/38 in melanocytes is essential for Tyrp1 trafficking remained unclear. In this study, we identified the residues of Rab32/38 (or Varp) that are critical for the formation of the Varp·Rab32/38 complex by performing Ala-based site-directed mutagenesis (FIGURE 1, FIGURE 5), and we evaluated the functional significance of the Varp-Rab32/38 interaction in Tyrp1 trafficking in melanocytes (FIGURE 3, FIGURE 8) by the knockdown-rescue approach. The results showed that neither Varp-binding-deficient Rab mutants (e.g. Rab38(V78A)) nor Rab32/38-binding-deficient Varp mutants (i.e. Varp(Q509A) and Varp(Y550A)) mediated the peripheral distribution of Tyrp1, whereas other Rab38 point mutants that possessed Varp-binding ability (e.g. Rab38(G73A) and Rab38(N74A)) and Varp point mutants with Rab32/38-binding ability (e.g. Varp(R557A)) behaved the same way as the respective wild-type protein in terms of the Tyrp1 distribution in melanocytes (FIGURE 3, FIGURE 8). These findings enabled us to conclude that the interaction between Rab32/38 and Varp in melanocytes is essential for the transport of Tyrp1 and for its peripheral distribution. We also demonstrated by the same knockdown-rescue approach that the VAMP7 binding activity of Varp, but not its Rab21-GEF activity, is essential for the peripheral melanosomal distribution of Tyrp1 in melanocytes (FIGURE 9, FIGURE 10). The requirement for the Rab21-GEF activity of Varp may be cell type-specific (neurons versus melanocytes) or cargo-specific (endosomal trafficking in neurites versus Tyrp1-containing vesicle trafficking), whereas VAMP7 binding activity is required for both neurite outgrowth and Tyrp1 transport. Because VAMP7 functions as a v-SNARE (42Chaineau M. Danglot L. Galli T. FEBS Lett. 2009; 583: 3817-3826Crossref PubMed Scopus (108) Google Scholar) and its partner t-SNARE syntaxin-3 has been shown to be localized on melanosomes (43Chi A. Valencia J.C. Hu Z.Z. Watabe H. Yamaguchi H. Mangini N.J. Huang H. Canfield V.A. Cheng K.C. Yang F. Abe R. Yamagishi S. Shabanowitz J. Hearing V.J. Wu C. Appella E. Hunt D.F. J. Proteome Res. 2006; 5: 3135-3144Crossref PubMed Scopus (150) Google Scholar), it is tempting to speculate that the VAMP7-syntaxin-3 interaction is involved in the fusion of Tyrp1-containing vesicles with melanosomes. Another suspected function of VAMP7 is as a bridge between the Varp·Rab32/38 complex and the adaptor complex AP-3, which is required for tyrosinase transport (44Höning S. Sandoval I.V. von Figura K. EMBO J. 1998; 17: 1304-1314Crossref PubMed Scopus (244) Google Scholar), because the Longin domain of VAMP7 interacts with AP-3 (45Martinez-Arca S. Rudge R. Vacca M. Raposo G. Camonis J. Proux-Gillardeaux V. Daviet L. Formstecher E. Hamburger A. Filippini F. D'Esposito M. Galli T. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9011-9016Crossref PubMed Scopus (161) Google Scholar). Further work will be necessary to determine the mechanism(s) by which the Varp·VAMP7 complex regulates Tyrp1 transport at the molecular level.Another important but unexpected finding in this study was that the level of expression of Varp in melanocytes is a key factor in the quality control of Tyrp1 in melanocytes. We found that either knockdown of Varp or overexpression of Varp alone in melanocytes caused a dramatic reduction in Tyrp1 signals (Fig. 7 and supplemental Fig. S5B). Interestingly, however, co-expression of Varp with Rab38 in melanocytes had no effect on Tyrp1 distribution or signals, suggesting that Varp free of Rab32/38 negatively regulates Tyrp1 trafficking, possibly by a proteasome-mediated degradation of Tyrp1 (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar) by unknown mechanisms, because the proteasome inhibitor MG132 greatly attenuated the reduction in Tyrp1 signals induced by overexpression of Varp (data not shown). As it has recently been reported that tyrosinase is degraded by endoplasmic reticulum-associated degradation (46Watabe H. Valencia J.C. Yasumoto K. Kushimoto T. Ando H. Muller J. Vieira W.D. Mizoguchi M. Appella E. Hearing V.J. J. Biol. Chem. 2004; 279: 7971-7981Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 47Ando H. Watabe H. Valencia J.C. Yasumoto K. Furumura M. Funasaka Y. Oka M. Ichihashi M. Hearing V.J. J. Biol. Chem. 2004; 279: 15427-15433Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 48Kageyama A. Oka M. Okada T. Nakamura S. Ueyama T. Saito N. Hearing V.J. Ichihashi M. Nishigori C. J. Biol. Chem. 2004; 279: 27774-27780Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and that Rab32/38 is also involved in tyrosinase trafficking (17Wasmeier C. Romao M. Plowright L. Bennett D.C. Raposo G. Seabra M.C. J. Cell Biol. 2006; 175: 271-281Crossref PubMed Scopus (209) Google Scholar), a similar degradation mechanism may operate for Tyrp1. It would therefore be interesting to determine whether compounds that promote the endoplasmic reticulum-associated degradation of tyrosinase (49Ando H. Ichihashi M. Hearing V.J. Int. J. Mol. Sci. 2009; 10: 4428-4434Crossref PubMed Scopus (29) Google Scholar) affect the function of Varp. An attempt to determine whether Varp is also involved in the trafficking of other melanogenic enzymes, including tyrosinase, and their quality control is now under way in our laboratory.In summary, we performed a structure-function analysis of Varp by site-directed mutagenesis in combination with the knockdown-rescue approach in cultured melanocytes. Our findings clearly indicated that Varp functions as a Rab32/38 effector in melanocytes and regulates Tyrp1 trafficking to melanosomes and that VAMP7 binding activity, but not Rab21-GEF activity, is also required for this process. The results further indicate that Varp is a key regulator of the quality control of Tyrp1 in melanocytes. IntroductionPigmentation of mammalian hair and skin requires proper formation and transport of melanosomes, one of the lysosome-related organelles that specifically synthesize and store melanin pigments, in melanocytes (reviewed in Refs. 1Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (348) Google Scholar, 2Raposo G. Marks M.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 786-797Crossref PubMed Scopus (364) Google Scholar). Defects in the formation and/or transport of melanosomes often cause pigmentary disorders, e.g. albinism and pigmentary dilution, in mammals (reviewed in Ref. 3Tomita Y. Suzuki T. Am. J. Med. Genet. 2004; 131C: 75-81Crossref PubMed Scopus (120) Google Scholar). The formation and transport of melanosomes involve a variety of intracellular membrane trafficking events, and several distinct Rab-type small GTPases, which are conserved membrane trafficking proteins in all eukaryotic cells (reviewed in Refs. 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 5Fukuda M. Cell. Mol. Life Sci. 2008; 65: 2801-2813Crossref PubMed Scopus (301) Google Scholar, 6Stenmark H. Nat. Rev. Mol. Cell Biol. 2009; 10: 513-525Crossref PubMed Scopus (2221) Google Scholar), have been shown to regulate the maturation and transport of melanosomes in mammalian epidermal melanocytes.The best characterized Rab isoform that is abundant on mature melanosomes in melanocytes is Rab27A (7Hume 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 (272) Google Scholar, 8Bahadoran P. Aberdam E. Mantoux F. Buscà 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 (171) Google Scholar, 9Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer 3rd, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). Rab27A regulates actin-based melanosome transport through interaction with its specific effector, Slac2-a/melanophilin (10Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 11Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer 3rd, J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (378) Google Scholar, 12Strom 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 (252) Google Scholar), and melanosome anchoring to the plasma membrane through interaction with another effector, Slp2-a (13Kuroda T.S. Fukuda M. Nat. Cell Biol. 2004; 6: 1195-1203Crossref PubMed Scopus (131) Google Scholar). As a result, Rab27A deficiency causes human type 2 Griscelli syndrome, which is characterized by silvery hair (i.e. partial albinism) (reviewed in Ref. 14Van Gele M. Dynoodt P. Lambert J. Pigment Cell Melanoma Res. 2009; 22: 268-282Crossref PubMed Scopus (116) Google Scholar and references therein). Two other Rab isoforms, Rab32 and Rab38, regulate an early step in melanogenesis, i.e. the transport of melanogenic enzymes to melanosomes (15Loftus S.K. Larson D.M. Baxter L.L. Antonellis A. Chen Y. Wu X. Jiang Y. Bittner M. Hammer 3rd, J.A. Pavan W.J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 4471-4476Crossref PubMed Scopus (138) Google Scholar, 16Park M. Serpinskaya A.S. Papalopulu N. Gelfand V.I. Curr. Biol. 2007; 17: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Wasmeier C. Romao M. Plowright L. Bennett D.C. Raposo G. Seabra M.C. J. Cell Biol. 2006; 175: 271-281Crossref PubMed Scopus (209) Google Scholar). Actually, dysfunction of Rab38 causes the diluted coat color of chocolate mice, presumably because of impairment of the targeting of tyrosinase-related protein 1 (Tyrp1) to melanosomes (15Loftus S.K. Larson D.M. Baxter L.L. Antonellis A. Chen Y. Wu X. Jiang Y. Bittner M. Hammer 3rd, J.A. Pavan W.J. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 4471-4476Crossref PubMed Scopus (138) Google Scholar). The discovery of the Rab32/38-specific binding protein Varp 4The abbreviations used are: Varp, VPS9-ankyrin-repeat protein; ANK, ankyrin; EGFP, enhanced green fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate. (VPS9-ankyrin-repeat protein; official symbol in the National Center for Biotechnology Information is Ankrd27) has recently been reported (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar, 19Wang F. Zhang H. Zhang X. Wang Y. Ren F. Zhang X. Zhai Y. Chang Z. Biochem. Biophys. Res. Commun. 2008; 372: 162-167Crossref PubMed Scopus (30) Google Scholar), and as its name indicates, Varp protein contains an N-terminal VPS9 (vacuolar protein sorting 9) domain and C-terminal tandem ankyrin repeat domains (named ANKR1 and ANKR2). The Varp VPS9 domain possesses Rab21-GEF (guanine nucleotide exchange factor) activity (20Zhang X. He X. Fu X.Y. Chang Z. J. Cell Sci. 2006; 119: 1053-1062Crossref PubMed Scopus (77) Google Scholar), and the ANKR1 domain functions as a specific GTP-Rab32/38-binding site (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar, 19Wang F. Zhang H. Zhang X. Wang Y. Ren F. Zhang X. Zhai Y. Chang Z. Biochem. Biophys. Res. Commun. 2008; 372: 162-167Crossref PubMed Scopus (30) Google Scholar). Even more recently, VAMP7/TI-VAMP (vesicle-associated membrane protein) has been shown to interact with the region between the ANKR1 domain and the ANKR2 domain and to regulate the neurite outgrowth of hippocampal neurons (21Burgo A. Sotirakis E. Simmler M.C. Verraes A. Chamot C. Simpson J.C. Lanzetti L. Proux-Gillardeaux V. Galli T. EMBO Rep. 2009; 10: 1117-1124Crossref PubMed Scopus (73) Google Scholar), but whether the Varp·VAMP7 complex is involved in melanogenesis has never been demonstrated. Because knockdown of Varp or overexpression of the ANKR1 domain, i.e. Rab32/38-binding site, in cultured melanocytes causes the disappearance of Tyrp1 signals from peripheral melanosomes (18Tamura K. Ohbayashi N. Maruta Y. Kanno E. Itoh T. Fukuda M. Mol. Biol. Cell. 2009; 20: 2900-2908Crossref PubMed Scopus (85) Google Scholar), Varp is likely to be involved in trafficking of Tyrp1 in concert with Rab32/38. However, the functional interaction between Rab32/38 and Varp (or VAMP7 and Varp) and the involvement of Rab21-GEF activity in Tyrp1 trafficking are not fully understood, and the structural basis of the molecular recognition of Rab32/38 by the Varp ANKR1 domain, including the residue(s) that are critical for Rab32/38 (or ANKR1) recognition, has never been elucidated.In this study, we identified critical residues for the Rab32/38-Varp interaction in Rab32/38 (or Varp) by performing Ala-based site-directed mutagenesis. A switch II mutant of Rab38, which carries a Val-to-Ala mutation at amino acid position 78 (named Rab38(V78A)), completely lost its Varp binding activity without any change in its intrinsic GTPase activity or subcellular localization. We showed that whereas wild-type Rab38 is able to restore the peripheral melanosomal localization of Tyrp1 in Rab32/38-deficient melanocytes, the Rab38(V78A) mutant is incapable of rescuing Tyrp1-trafficking deficiency and that ANKR1 point mutants, Varp(Q509A) and Varp(Y550A), are also unable to rescue Tyrp1 deficiency in Varp-deficient melanocytes. We then investigated two Varp mutants carrying a point mutation in the VPS9 domain and a Varp deletion mutant lacking the VAMP7-binding site (named ΔVID) and showed that VAMP7 binding to Varp is required for Tyrp1 trafficking, but that Rab21-GEF activity is not. We discuss the structure-function relationships of Varp in Tyrp1 trafficking to melanosomes in melanocytes on the basis of these findings.
Referência(s)