Kuroda Taruho S, Itoh Takashi, Fukuda Mitsunori
Methods Enzymol. 2005;403:419-31. doi: 10.1016/S0076-6879(05)03037-5.
Slac2-a/melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.
Slac2-a/黑素亲和蛋白通过连接黑素小体结合的Rab27A和一种基于肌动蛋白的运动蛋白肌球蛋白Va，来调节哺乳动物皮肤黑素细胞中的黑素小体运输。Slac2-a由一个N端的Slp同源结构域（SHD）、一个位于中间区域的肌球蛋白Va结合结构域（MBD）和一个位于C端的肌动蛋白结合结构域（ABD）组成。其中，SHD已被确定为特定的GTP-Rab27结合结构域。Slac2-a/mlph基因的突变会导致人类III型格里塞利综合征和铅灰小鼠出现色素沉着异常（即黑素细胞中核周黑素小体聚集），原因是无法形成由Rab27A、Slac2-a和肌球蛋白Va组成的三方蛋白复合物。在本章中，我们描述了一些方法，包括结合显性负性方法和RNA干扰技术的体内黑素小体分布测定法，这些方法已被用于分析Slac2-a在黑素细胞黑素小体运输中的功能。
