Studies on multifunctional Properties of Tetrahymena citrate synthase

四膜虫14 nm细丝蛋白/柠檬酸合成酶(14FP)是一种具有细胞骨架和柠檬酸合成酶功能的双功能蛋白。在本研究中，我对14FP的双功能调控系统进行了研究。(1)在大肠杆菌中表达、纯化和鉴定了重组14FP。重组的14FP具有柠檬酸合成酶活性，并形成了14 nm的细丝。这些结果表明，14FP本身具有双功能。(2)双向凝胶电泳法分析表明，从四膜虫线粒体中纯化的柠檬酸合成酶含有PI。7.7和8.0异构体，而组装和拆卸过程中纯化的14 nm细丝蛋白含有PI。7.7、8.0和8.4亚型。圆周率。7.7和/或PI。8.0亚型显示柠檬酸合成酶活性，但等电点。8.4异构体不具有这种活性。这些结果表明，这些异构体的多态是由翻译后修饰引起的。(3)所有异构体都是磷酸…由于它们之间的磷酸化程度的不同，其亚型的甲基化程度和多态程度更高。此外，去磷酸化导致柠檬酸合成酶活性下降，激活了14FP的成丝能力。这些结果表明，磷酸化和去磷酸化调节14FP的双功能特性。(4)14FP亲和层析筛选的14FP结合蛋白表明，四膜虫线粒体伴侣蛋白Hsp60和mthsp70以一种依赖于ATP的方式与14FP结合。免疫荧光显微镜显示四膜虫Hsp60定位于线粒体，可能参与了包括14FP在内的线粒体蛋白的折叠。此外，四膜虫Hsp60还存在于线粒体外部位，包括基体和口腔器官，尤其是在细胞分裂过程中发育的口腔器官。由于14FP参与了口腔的形态发生，所以四膜虫Hsp60可能参与了口腔的形态发生并调节了14FP的双功能特性。较少

Tetrahymena 14-nm filament protein/citrate synthase (14FP) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase. In this study, I examined regulation systems of bifunctional properties of 14FP.(1) A recombinant 14FP was expressed in E. coli, purified and characterized. The recombinant 14FP has the citrate synthase activity and formed 14-nm filaments. These results suggest that 14FP itself is bifunctional.(2) Analysis by two-dimensional gel electrophoresis demonstrated that the citrate synthase purified from Tetrahymena mitochondria comprised pI. 7.7 and 8.0 isoforms, while the 14-nm filament protein purified by the assembly and disassembly procedures comprised pI. 7.7, 8.0 and 8.4 isoforms. The pI. 7.7 and/or pI. 8.0 isoforms exhibit the citrate synthase activity, but the pI. 8.4 isoform did not possess this activity. These results suggest that the polymorphism of these isoforms is caused by some posttranslational modifications.(3) All isoforms were phosph … More orylated, and polymorphism of its isoforms resulted from differences in the degree of phosphorylation between them. Moreover, dephosphorylation led to a decrease in citrate synthase activity and activated filament-forming ability of 14FP. These results suggest that phosphorylation and dephosphorylation regulate the bifunctional properties of 14FP.(4) The screening of 14FP-binding proteins using 14FP-affinity chromatography demonstrated that Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, bound to 14FP in an ATP dependent manner. Immunofluorescence microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria, possibly participating in the folding of mitochondrial proteins including 14FP. Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies and oral apparatus, especially at developing oral apparatus during cell division. Since 14FP participates in the oral morphogenesis, it is possible that Tetrahymena hsp60 is involved in the oral morphogenesis and regulates the bifunctional properties of 14FP. Less