Nagashima Kazuaki, Torii Seiji, Yi Zhaohong, Igarashi Michihiro, Okamoto Koichi, Takeuchi Toshiyuki, Izumi Tetsuro
Laboratory of Gene Engineering, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, Japan.
FEBS Lett. 2002 Apr 24;517(1-3):233-8. doi: 10.1016/s0014-5793(02)02634-0.
Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVa(d)), ashen (Rab27a(ash)), and leaden (Mlph(ln)), act in the same or overlapping pathways. Here we have cloned and characterized a human melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimentation assays using recombinant proteins reveal that melanophilin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respectively. Moreover, we show that Rab27a, melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/granuphilin-like Rab effector family constitutes an alpha-helix-based coiled-coil structure.
Rab鸟苷三磷酸酶（Rab GTPases）通过招募其特定的效应蛋白来调节膜运输途径。黑色素亲和蛋白（Melanophilin）是一种假定的Rab效应蛋白，最近被鉴定为在铅灰小鼠中发生突变的基因，在这种小鼠中，黑素细胞中黑素体的外周定位受损。遗传学研究表明，三个毛色突变基因，即稀释基因（dilute，MyoVa(d)）、灰白色基因（ashen，Rab27a(ash)）和铅灰基因（leaden，Mlph(ln)），在相同或重叠的途径中起作用。在这里，我们克隆并鉴定了一种人类黑色素亲和蛋白同源物，它属于rabphilin3/颗粒亲和蛋白样Rab效应蛋白家族。使用重组蛋白进行的共沉降分析表明，黑色素亲和蛋白分别通过其N端和第一个C端卷曲螺旋区域直接与Rab27a和肌球蛋白Va结合。此外，我们表明Rab27a、黑色素亲和蛋白和肌球蛋白Va在人类黑素细胞系HMV-II中形成三元复合物。这些发现表明，在黑素体运输过程中，黑色素亲和蛋白在连接黑素体上的Rab27a和肌动蛋白丝上的肌球蛋白Va方面发挥作用。我们还提出，在一个新的rabphilin3/颗粒亲和蛋白样Rab效应蛋白家族中保守的Rab结合区域构成了一种基于α螺旋的卷曲螺旋结构。
