Mutation of wrb, a Component of the Guided Entry of Tail-Anchored Protein Pathway, Disrupts Photoreceptor Synapse Structure and Function.
Mutation of wrb, a Component of the Guided Entry of Tail-Anchored Protein Pathway, Disrupts Photoreceptor Synapse Structure and Function.
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DOI：
10.1167/iovs.15-18996
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发表时间：
2016-06-01
影响因子：
4.4
通讯作者：
Perkins BD
中科院分区：
文献类型：
--
作者：
Daniele LL;Emran F;Lobo GP;Gaivin RJ;Perkins BD
尾部锚定(Tail-Anchored，TA)蛋白在C末端含有一个疏水结构域，通过GET(Tail-Anchored Proteins，引导进入尾部锚定蛋白)途径被翻译后插入内质网。GET通路在光感受器中的作用尚不清楚。这项研究的目的是描述斑马鱼弹球巫师突变体的特征，该突变体破坏了GET途径的核心成分WRB。通过视网膜电图术、视动反应测量(OKR)、免疫组织化学和电子显微镜分析，研究了5日龄斑马鱼幼体的带状突触功能、蛋白质表达和超微结构。实时荧光定量聚合酶链式反应和原位杂交检测WrB基因的表达。WRB基因突变使OKR消失，ERG b波显著降低，但a波无明显变化。Rbeye和SV2在Wrb突变体的光感受器和毛细胞中部分错位。在Wrb−/−突变体中，光感受器与双极细胞之间的接触较少。WRB在整个神经系统都有表达，WRB定位于光感受器的内质网和突触区域。靶向内质网TA蛋白的胞浆ATPase trc40的吗啡敲除也降低了OKR。Wrb的过表达完全恢复了突变体的对比敏感度，而不能结合Trc40的突变体WrbR73A的过表达则没有。蛋白质WRB和Trc40是光感受器和双极细胞之间突触传递所必需的，这表明通过TRC途径插入TA蛋白是带状突触组装和功能的关键步骤。
Tail-anchored (TA) proteins contain a single hydrophobic domain at the C-terminus and are posttranslationally inserted into the ER membrane via the GET (guided entry of tail-anchored proteins) pathway. The role of the GET pathway in photoreceptors is unexplored. The goal of this study was to characterize the zebrafish pinball wizard mutant, which disrupts Wrb, a core component of the GET pathway. Electroretinography, optokinetic response measurements (OKR), immunohistochemistry, and electron microscopy analyses were employed to assess ribbon synapse function, protein expression, and ultrastructure in 5-day-old zebrafish larvae. Expression of wrb was investigated with real-time qRT-PCR and in situ hybridization. Mutation of wrb abolished the OKR and greatly diminished the ERG b-wave, but not the a-wave. Ribeye and SV2 were partially mislocalized in both photoreceptors and hair cells of wrb mutants. Fewer contacts were seen between photoreceptors and bipolar cells in wrb−/− mutants. Expression of wrb was observed throughout the nervous system and Wrb localized to the ER and synaptic region of photoreceptors. Morpholino knockdown of the cytosolic ATPase trc40, which targets TA proteins to the ER, also diminished the OKR. Overexpression of wrb fully restored contrast sensitivity in mutants, while overexpression of mutant wrbR73A, which cannot bind Trc40, did not. Proteins Wrb and Trc40 are required for synaptic transmission between photoreceptors and bipolar cells, indicating that TA protein insertion by the TRC pathway is a critical step in ribbon synapse assembly and function.