Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli syndrome
Characterization of the molecular defects in Rab27a, caused by RAB27A missense mutations found in patients with Griscelli syndrome
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DOI：
10.1074/jbc.m211996200
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发表时间：
2003-03-28
影响因子：
4.8
通讯作者：
Ballotti, R
中科院分区：
文献类型：
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作者：
Bahadoran, P;Busca, R;Ballotti, R
Rab 27 a在黑素体向黑素细胞树突尖端的转运中起关键作用，并且Rab 27 A的突变损害黑素体转运，导致在几个Griscelli综合征（GS）患者中发现的色素稀释和免疫缺陷。有趣的是，三名GS患者在RAB 27 A中存在单一纯合错义突变，导致W73 G，L130 P和A152 P转换，影响Rab蛋白中高度保守的残基。然而，这些突变的功能后果尚未研究。在本报告中，我们评估了这些突变体的过度表达对黑素体，亲黑素蛋白和肌球蛋白-Va在B16黑色素瘤细胞中的定位的影响。然后，我们研究了Rab 27 a功能的几个关键参数，包括GTP结合和与黑素蛋白/肌球蛋白-Va复合物的相互作用，该复合物将黑素体连接到肌动蛋白网络。我们的结果表明Rab 27 a-L130 P不能结合GTP，不与亲黑素蛋白相互作用，因此不能允许黑素体在肌动蛋白丝上转运。有趣的是，Rab 27 a-W73 G结合GTP，但不与亲黑素相互作用。因此，Rab 27 a-W73 G不能支持肌动蛋白依赖的黑素体转运。最后，Rab 27 a-A152 P结合GTP和黑素蛋白。然而，Rab 27 a-A152 P不允许黑素体转运，并且作为显性负突变体，因为其在B16黑色素瘤细胞中的过表达模拟GS表型。因此，Rab 27 a与黑素/肌球蛋白-Va的相互作用不足以确保正确的黑素体转运。我们的研究结果指出了Rab 27 a功能的意外复杂性，并为寻找控制Rab 27 a依赖性囊泡转运的新Rab 27 a效应子或调节子开辟了道路。
Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.