title: Rab27a and MyoVa are the primary Mlph interactors regulating melanosome transport in melanocytes.
abstract: Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect Mlph-MyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.
authors: Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ, UK. a.hume@imperial.ac.uk
authors: Alistair N
authors: AN
authors: Hume
authors: Dmitry S
authors: DS
authors: Ushakov
authors: Abul K
authors: AK
authors: Tarafder
authors: Michael A
authors: MA
authors: Ferenczi
authors: Miguel C
authors: MC
authors: Seabra
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C405804:EB1 microtubule binding proteins; C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; C435724:Mlph protein, mouse; C513082:Myo5a protein, mouse; D034741:RNA, Small Interfering; D000075745:rab27 GTP-Binding Proteins; D024701:Myosin Type V; C512962:Rab27a protein, mouse; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins