Functional analysis of Slac2-c/MyRIP as a linker protein between melanosomes and myosin VIIa
Functional analysis of Slac2-c/MyRIP as a linker protein between melanosomes and myosin VIIa
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DOI：
10.1074/jbc.m501465200
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发表时间：
2005-07-29
影响因子：
4.8
通讯作者：
Fukuda, M
中科院分区：
文献类型：
--
作者：
Kuroda, TS;Fukuda, M
Slac 2-c/MyRIP是一种体外Rab 27 A和肌球蛋白Va/VIIa结合蛋白，最近被提出通过直接连接黑素小体结合的Rab 27 A和肌球蛋白VIIa来调节视网膜色素上皮细胞中的视网膜黑素小体转运;然而，Slac 2-c在黑素小体转运中的确切功能从未被阐明。在这项研究中，我们使用皮肤黑素细胞中的黑素体转运作为视网膜黑素体转运的模型，并通过Slac 2-c与肌球蛋白VIIa在Slac 2-a耗尽的黑素细胞中的异位表达来分析Slac 2-c在黑素体转运中的体内功能。体外结合实验表明，与肌球蛋白Va的结合亲和力相比，肌球蛋白VIIa对Slac 2-c具有更大的亲和力，并且Slac 2-c的肌球蛋白VIIa结合结构域不同于先前表征的在Slac 2-a/黑素蛋白和Slac 2-c之间保守的肌球蛋白Va结合结构域。与此结果一致，在黑素细胞中表达的青色荧光蛋白标记的Slac 2-c通过与Rab 27 A的特异性相互作用定位在黑素体上，并将共表达的黄色荧光蛋白标记的肌球蛋白VIIa募集到黑素体上，而不干扰正常的外周黑素体分布，而当肌球蛋白VIIa单独在黑素细胞中表达时，它不定位在黑素体上。此外，在黑素细胞中异位表达的Slac 2-c并没有挽救由用特异性小干扰RNA敲低内源性Slac 2-a诱导的核周聚集表型，而Slac 2-c.肌球蛋白VIIa复合物的表达支持Slac 2-a耗尽的黑素细胞中的正常黑素体分布，表明Slac 2-c在黑素体转运中作为肌球蛋白VIIa受体而不是肌球蛋白Va受体起作用。基于这些发现，我们提出，Slac 2-c作为一个功能性的肌球蛋白VIIa受体和Rab27A.Slac2-c.肌球蛋白VIIa三方蛋白复合物调节色素上皮细胞中视网膜黑素体的运输。
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.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.myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.