RanGTPase激活蛋白RanGAP在嗜热四膜虫细胞中的定位及功能

Localization and Function of RanGTPase Activating Protein(RanGAP)from Tetrahymena thermophila

RanGTPase激活蛋白（RanGTPase-activating protein，RanGAP）和Ran相互作用，提高了RanGTPase水解GTP的效率．RanGAP参与细胞内核质运输、纺锤体组装、核膜重建和异染色质的组装．生物进化过程中，不同生物的RanGAP表现出结构和功能的多样性．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的RanGTPase激活蛋白基因RanGAP（TTHERM_00766430）．实时荧光定量PCR表明，RanGAP在四膜虫营养生长、饥饿和有性生殖过程中均有表达，且在有性生殖4～6h表达水平最高．免疫荧光定位表明，在营养生长期、饥饿期及有性生殖的早期，RanGAP定位于细胞质中；在有性生殖后期，RanGAP定位于凋亡的大核中．过表达RanGAP的细胞增殖速率下降，大核分裂和胞质缢缩异常，产生无大核细胞．敲减RanGAP的细胞大核形态异常，细胞增殖速率下降，无丝分裂受到抑制，进而产生无大核细胞．RanGAP的过表达或敲除分别引起四膜虫RAN1，RanBP1和RCC1基因的表达下调或上调．结果表明，RanGAP通过Ran信号通路调控了嗜热四膜虫无性生殖过程中大核的无丝分裂，并可能参与了有性生殖过程中亲本大核的凋亡．

RanGTPase activating protein （RanGAP） interacts with Ran to increase the hydrolysis rate of GTPs bound to Ran proteins. Although diversified in different organisms, functionally conserved RanGAPs participate in the processes of nucleocytoplasmic transport, mitotic spindle formation, nuclear envelope assembly and heterochromatin assembly. In this study, we identified a RanGAP （TTHERM 00766430） from Tetrahymena thermophila, which expressed at the vegetative growth, starvation and conjugation conditions as determined by real-time PCR, particularly peaked at 4-6 hours during conjugation stage. HA-RanGAP was found to localize in the cytoplasm in various stages, and was detected in the apoptotic parental macronuclei in late conjugation stage. Either overexpression or knockdown of RanGAP inhibited cell proliferation, led to abnormal macronuclear amitosis and cytokinesis, and eventually produced a macronuclear cells. Meanwhile, RanGAP overexpression resulted in down-regulation of RAN1, RanBP1 and RCC1 in Tetrahymena, while RanGAP knockdown did the contrary. The results showed that RanGAP regulated the macronuclear amitosis and involved in the parental macronuclear apoptosis in Tetrahymena.