Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella
Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella
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DOI：
10.1074/jbc.m111.241760
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发表时间：
2011-08-19
影响因子：
4.8
通讯作者：
Nicastro, Daniela
中科院分区：
文献类型：
--
作者：
Lin, Jianfeng;Tritschler, Douglas;Nicastro, Daniela
运动的纤毛和鞭毛产生的定向流动对于许多过程至关重要，包括人类发育和器官功能。正常的搏动需要成千上万的动力蛋白马达的控制和协调，而连接蛋白-动力蛋白调节复合物（N-DRC）已被确定为协调动力蛋白活动的重要调节节点。连接蛋白连接似乎是动力蛋白驱动的线性微管滑动到鞭毛弯曲的转变的关键，但N-DRC的分子组成和机制仍然在很大程度上未知。在这里，我们使用蛋白质组学，特别注意蛋白质磷酸化，以分析的N-DRC的组成，并确定哪些亚基可能是重要的信号转导。二维电泳和MALDI-TOF质谱的野生型和突变体鞭毛轴丝衣原体确定了12个N-DRC相关的蛋白质，包括所有7个以前观察到的N-DRC组件。序列和PCR分析确定的突变负责的sup-pf-4菌株的表型，和生化比较与径向辐条突变体揭示了两个组件，可能连接的N-DRC和径向辐条。磷酸化蛋白质组学揭示了8种蛋白质的磷酸化亚型，其亚型模式随基因型而变化，以及两种组分可能通过其磷酸化状态在N-DRC功能中发挥关键作用。这些数据被组装成N-DRC的模型，解释了其调节功能的各个方面。
The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.