Functional Analysis of Slac2-c/MyRIP as a Linker Protein between Melanosomes and Myosin VIIa*[boxs

Taruho S Kuroda,Mitsunori Fukuda

doi:10.1074/jbc.m501465200

Abstract

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c x myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A.Slac2-c x myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Highlights

Rab27A is a member of the small GTPase Rab family and has recently been identified as a critical regulator of various types of membrane trafficking, including vesicle exocytosis in some secretory cells and melanosome transport in melanocytes
Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes
Since we previously showed that a conserved acidic cluster in the myosinbinding domain (MBD) of Slac2-a is required for myosin Va binding activity [12], we prepared a similar mutant of Slac2-c and tested its myosin Va/VIIa-tail binding activity

Summary

Introduction

Rab27A is a member of the small GTPase Rab family (reviewed in Refs. 1 and 2) and has recently been identified as a critical regulator of various types of membrane trafficking, including vesicle exocytosis in some secretory cells and melanosome transport in melanocytes (reviewed in Refs. 3–5). We propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A1⁄7Slac2-c1⁄7myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Results

Conclusion