Chlamydomonas ODA10 is a conserved axonemal protein that plays a unique role in outer dynein arm assembly.
Chlamydomonas ODA10 is a conserved axonemal protein that plays a unique role in outer dynein arm assembly.
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DOI：
10.1091/mbc.e13-06-0310
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发表时间：
2013-12
影响因子：
3.3
通讯作者：
Mitchell DR
中科院分区：
文献类型：
--
作者：
Dean AB;Mitchell DR
在体内，轴丝动力蛋白组装成纤毛是一个多步骤的过程。我们发现，衣原体ODA10编码的轴丝蛋白所需的外臂动力蛋白装配的后期步骤。一旦动力蛋白臂组装好，它们就可以被提取出来，而ODA10 p蛋白不再需要高亲和力地结合到轴丝结合位点上。外动力蛋白臂（ODA）的组装需要多个步骤，并且除了动力蛋白亚基之外还涉及多个蛋白质。衣原体ODA10，ODA5，和ODA8基因座遗传相互作用，并假设作为一个轴丝附件复合体的功能，但只有ODA5p以前的特点。我们定位克隆了ODA 10，并通过用血凝素标记的cDNA拯救ODA 10突变体来鉴定该基因。ODA10序列预测了与小鼠ccdc 151同源的保守卷曲螺旋蛋白。ODA 10 p存在于细胞质和鞭毛中，在去污剂处理后保持轴丝，并用0.6 M NaCl提取。外臂动力蛋白和ODA10 p反弹的轴丝时，脱盐提取物与oda10突变体轴丝混合。这些提取物的蔗糖梯度分离显示，ODA 10 p在梯度顶部附近沉积，而不是与23S外动力蛋白臂蛋白。出乎意料的是，动力蛋白和ODA10 p组分能够单独结合oda10轴丝。ODA10p以野生型水平存在于oda8突变体鞭毛上。然而，ODA 10 p不能组装到oda5鞭毛中，并且不存在于oda5细胞质中，这表明ODA 10 p在体内的稳定性需要ODA 5 p。结果表明，ODA10 p不作为传统定义的对接复合物的一部分发挥作用。
In vivo, assembly of axonemal dyneins into cilia is a multi-step process. We show that Chlamydomonas ODA10 encodes an axonemal protein required for a late step in outer arm dynein assembly. Once dynein arms have assembled, they can be extracted and the ODA10p protein is no longer required for high-affinity binding onto axonemal binding sites. Assembly of outer dynein arms (ODAs) requires multiple steps and involves multiple proteins in addition to dynein subunits. The Chlamydomonas ODA10, ODA5, and ODA8 loci genetically interact and are hypothesized to function as an axonemal accessory complex, but only ODA5p was previously characterized. We positionally cloned ODA10 and identified the gene by rescuing an oda10 mutant with a hemagglutinin-tagged cDNA. ODA10 sequence predicts a conserved coiled-coil protein homologous to mouse ccdc151. ODA10p is present in cytoplasm and flagella, remains axonemal after detergent treatment, and is extracted with 0.6 M NaCl. Both outer arm dynein and ODA10p rebound to the axonemes when desalted extracts are mixed with oda10-mutant axonemes. Sucrose gradient separation of these extracts shows that ODA10p sediments near the top of the gradient, not with 23S outer dynein arm proteins. Unexpectedly, dynein and ODA10p fractions are able to bind individually to oda10 axonemes. ODA10p is present on oda8-mutant flagella at wild-type levels. However, ODA10p does not assemble into oda5 flagella and is absent from oda5 cytoplasm, suggesting a necessity of ODA5p for stability of ODA10p in vivo. The results suggest that ODA10p does not function as a part of a traditionally defined docking complex.