Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes
Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes
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DOI：
10.1091/mbc.e08-12-1161
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发表时间：
2009-06-15
影响因子：
3.3
通讯作者：
Fukuda, Mitsunori
中科院分区：
文献类型：
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作者：
Tamura, Kanako;Ohbayashi, Norihiko;Fukuda, Mitsunori
两个小的GTPase Rabs， Rab32和Rab38，最近被提出在哺乳动物表皮黑色素细胞中调节黑色素生成酶到黑色素小体的运输；然而，rab32 /38介导的黑素生成酶运输的确切分子机制尚未明确，因为尚未发现rab32 /38特异性效应物。在本研究中，我们以鸟苷三磷酸（GTP）锁定的Rab32/38为诱饵，通过酵母双杂交实验筛选Rab32/38特异性效应物，发现vps9 -锚蛋白重复蛋白(Varp)/Ankrd27作为Rab21的鸟嘌呤核苷酸交换因子（GEF），在小鼠黑素细胞系melana中作为Rab32/38特异性结合蛋白发挥作用。缺失分析表明，第一锚蛋白重复序列（ANKR1）结构域作为gtp依赖的rab32 /38结合域发挥作用，而n端VPS9结构域（即Rab21-GEF结构域）不具有这种功能。在melana细胞中，小干扰rna介导的内源性Varp的敲低导致黑素体中Tyrp1（酪氨酸酶相关蛋白1）信号的显著减少，但未引起Pmel17信号的任何减少。此外，在melan-a细胞中，ANKR1结构域的表达也导致Tyrp1信号的显著减少，而VPS9结构域则没有影响。基于这些发现，我们提出Varp作为Rab32/38效应物在黑素细胞中控制Tyrp1的运输。
Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.