To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 μm) and full-length (~11 μm) axonemes of Chlamydomonas. Known compoents of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 μm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.

中文翻译：

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FAP93 在衣藻纤毛近端轴丝处的组装

为了鉴定近端睫状轴丝特异的蛋白质，我们使用 iTRAQ 比较了衣藻的短轴丝（~2 μm）和全长轴丝（~11 μm） 。近端轴丝的已知成分，如次要动力蛋白重链和 LF5 激酶以及纤毛尖端蛋白 FAP256 (CEP104) 和 EB1 在短轴丝中富集，而沿轴丝长度存在的蛋白质在两个样品中丰度相似。iTRAQ 分析显示，功能未知的蛋白质 FAP93 和蛋白磷酸酶 2A (PP2A) 在短轴丝中富集。一致地，免疫印迹显示 FAP93 和 PP2A 在短轴丝中富集，免疫荧光证实了 FAP93 的定位和 PP2A 在近端轴丝中的富集。纤毛再生表明，FAP93 连续组装，但比其他轴丝结构更慢，并在稳态轴丝中终止于 1.03 μm。FAP93 组装的长度与纤毛长度相关，表明 FAP93 的组装依赖于纤毛长度。双核拯救实验表明，FAP93 可以独立于 IFT 运输进行组装。此外，GFP 标记的 FAP93 的 FRAP 分析表明 FAP93 稳定锚定在轴丝中。FAP93 可以作为在近端轴丝组装其他特定蛋白质的支架。