Ahn Hye-Jin, Kim Sehra, Nam Ho-Woo
Department of Parasitology and the Catholic Institute of Parasitic Diseases, College of Medicine, Catholic University of Korea, Seoul 137-701, Korea.
Korean J Parasitol. 2007 Sep;45(3):165-74. doi: 10.3347/kjp.2007.45.3.165.
Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
在HeLa细胞中作为绿色荧光蛋白-GRA10融合蛋白表达的刚地弓形虫GRA10迅速且完全地转移至细胞核内的核仁。通过针对GRA10（Tg378）、B23（核磷蛋白）和C23（核仁素）的单克隆抗体，使绿色荧光蛋白-GRA10转染图像与免疫荧光图像重叠，从形态学上看，GRA10特异性地集中在核仁的致密纤维成分中。GRA10的核仁转位是由GRA10假定的核仁定位序列（NoLS）引起的。通过谷胱甘肽-S-转移酶下拉分析和免疫沉淀分析，在酵母双杂交技术中证实了GRA10与TATA结合蛋白相关因子1B（TAF1B）的相互作用。共转染后，GRA10和TAF1B也在核仁中共定位。放线菌素D影响GRA10的核仁凝聚。在低剂量放线菌素D作用下，表达的绿色荧光蛋白-GRA10均匀分布于核质中，核仁位置在核质中仍呈空洞状。GRA10的核仁定位及其与TAF1B的相互作用表明GRA10参与宿主细胞的核糖体RNA合成，以利于刚地弓形虫的寄生。
