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Vol. 17, Issue 11, 4720-4735, November 2006

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A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

Alistair N. Hume^*, Abul K. Tarafder^*, José S. Ramalho, Elena V. Sviderskaya, and Miguel C. Seabra^*

*Molecular and Cellular Medicine, Division of Biomedical Sciences, Imperial College London, London SW7 2AZ, United Kingdom; Centre of Ophthalmology, Biomedical Institute for Research in Light and Image, University of Coimbra, 3000 Coimbra, Portugal; and Department of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, United Kingdom

Submitted May 25, 2006; Accepted August 9, 2006
Monitoring Editor: Jennifer Lippincott-Schwartz

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0457) on August 16, 2006.

Address correspondence to: Miguel C. Seabra (m.seabra{at}imperial.ac.uk)

Abbreviations used: ABD, actin-binding domain; EB1, end-binding protein 1; EFBD, exon F-binding domain; GS, Griscelli syndrome; GTBD, globular tail-binding domain; ln, leaden; MBD, myosinVa–binding domain; Mlph, melanophilin; R27BD, Rab27a-binding domain; SHD, synaptotagmin homology domain; ZnF, Zn²⁺ finger; TRP1, tyrosinase-related protein 1; TRP2, tyrosinase-related protein 2.

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