Studies on multifunctional Properties of Tetrahymena citrate synthase

四膜虫14nm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双重功能的蛋白。在本研究中，我研究了14FP双功能特性的调控系统。(1)在大肠杆菌中表达了重组14FP，并进行了纯化和表征。重组14FP具有柠檬酸合酶活性，可形成14nm的丝状结构。这些结果表明14FP本身具有双重功能。(2)双向凝胶电泳分析表明，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，而通过组装和拆卸程序纯化的14 nm丝蛋白包含pI。7.7, 8.0和8.4异构体。π。7.7和/或pI。8.0同工型表现出柠檬酸合酶活性，而pI。8.4 isoform不具有这种活性。这些结果表明，这些异构体的多态性是由一些翻译后修饰引起的。(3)所有同工异构体均磷酸化程度较高，其多态性是由磷酸化程度不同引起的。此外，去磷酸化导致14FP的柠檬酸合成酶活性降低和成丝能力激活。这些结果表明，磷酸化和去磷酸化调节了14FP的双功能特性。(4)利用14FP亲和层析法筛选14FP结合蛋白，发现四膜虫线粒体伴侣蛋白hsp60和mthsp70以ATP依赖的方式与14FP结合。单克隆抗体免疫荧光显微镜显示，四膜虫hsp60定位于线粒体，可能参与线粒体蛋白的折叠，包括14FP。此外，四膜虫hsp60也存在于包括基体和口腔器官在内的线粒体外部位，特别是在细胞分裂过程中发育中的口腔器官。由于14FP参与了口腔形态发生，因此四膜虫hsp60可能参与了口腔形态发生并调控了14FP的双功能特性。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