嗜热四膜虫异染色质蛋白Tcd1磷酸化位点的突变分析

四膜虫异染色质蛋白Tcd1在有性生殖时期特异表达，在大核基因组重排以及修复过程中发挥作用。磷酸化蛋白质组学分析表明，Tcd1存在3个磷酸化位点：S301，S303和S535。然而，Tcd1磷酸化修饰与其功能的关系并不清楚。本研究对TCD1基因的3个磷酸化位点进行了模拟磷酸化和模拟去磷酸化定点突变，获得模拟磷酸化突变基因TCD1S301D (TCD1S1D)、TCD1S301DS303D (TCD1S2D)与TCD1S301DS303DS535D (TCD1S3D) 和模拟去磷酸化的突变基因TCD1S301A (TCD1S1A)、TCD1S301AS303A (TCD1S2A)与TCD1S301AS303AS535A (TCD1S3A)。分别构建了不同突变体的过表达载体，转化四膜虫细胞并筛选获得不同突变体细胞株。Western印迹分析表明，Tcd1S1D、Tcd1S2D、Tcd1S3D与Tcd1S1A、Tcd1S2A和Tcd1S3A在四膜虫有性生殖期表达。免疫荧光定位分析发现，Tcd1S1D点状定位于细胞质中，Tcd1S2D在有性生殖初期点状定位于细胞质中，在新大核上形成均匀的定位，Tcd1S3D无法定位于亲本大核上，只是均匀定位于新大核上。Tcd1S2A和Tcd1S3A在新大核形成异常的块状定位，并且与异染色质蛋白Pdd1不能共定位。结果表明，Tcd1不同位点的磷酸化和去磷酸化修饰的动态变化决定了其在四膜虫细胞中的定位模式。

Mutational analysis of phosphorylation site of Tcd1 from Tetrahymena thermophila

The Tetrahymena chromodomain protein (Tcd1) is specifically expressed in Tetrahymena thermophila during sexual reproduction and plays an important role in the rearrangement and repair of the macronuclear genome. Mass spectrometry-based global phosphoproteomic analysis showed that Tcd1 contains three phosphorylation sites: S301, S303 and S535. But the function of the phosphorylation modification of Tcd1 is unclear. In this study, we mutated these sites and constructed mutation plasmids: TCD1S301D (TCD1S1D), TCD1S301DS303D (TCD1S2D), TCD1 S301DS303DS535D (TCD1S3D), TCD1S301A (TCD1S1A), TCD1S301AS303A (TCD1S2A), and TCD1S301AS303AS535A (TCD1S3A). The plasmids were transformed into different mating types of Tetrahymena cells and the mutant cells were screened and identified. Western blotting showed that Tcd1S1D, Tcd1S2D, Tcd1S3D, Tcd1S1A, Tcd1S2A and Tcd1S3A were stably expressed during the conjugation stage. Immunofluorescence staining showed that punctate Tcd1S1D staining was discernable in the cytoplasm during the whole conjugation stage. Tcd1S2D and Tcd1S3D localized in the cytoplasm at the early sexual stage and relocalized in the new macronuclei at the late conjugation stage. Tcd1S1A, Tcd1S2A and Tcd1S3A localized in the parental macronuclei and new macronuclei. But Tcd1S2A and Tcd1S3A formed aberrant structures in the new macronuclei and failed to colocalize with Pdd1. These results suggested that dynamic phosphorylation and dephosphorylation modification of Tcd1 determines its localization pattern and function in Tetrahymena.