Multifunction of Tetrahymena citrate synthase

四膜虫14 nm丝状蛋白cDNA的克隆表明，该蛋白的一级结构与猪柠檬酸合成酶具有高度的序列同源性。为了验证这一假说，我们比较了纯化的柠檬酸合成酶和14 nm细丝蛋白的抗原性和酶活性特性。抗14 nm细丝蛋白抗体与柠檬酸合成酶发生交叉反应，抑制其酶活性。为了确定14 nm纤维蛋白基因和mRNA的数目，采用Southern杂交和北方杂交技术对14 nm纤维蛋白基因和mRNA进行了检测。这些结果表明，四膜虫只有单一类型的基因和单一类型的mRNA的14 nm的细丝蛋白。免疫电镜显示14 nm细丝蛋白/柠檬酸合成酶在线粒体基质中形成细丝束，并在线粒体基质中形成14 nm细丝蛋白。为了阐明丝状体形成与柠檬酸合成酶活性之间的关系，我们分析了离体条件下丝状体形成过程中柠檬酸合成酶活性的变化。14 nm丝状蛋白在聚合态时柠檬酸合成酶活性较低，而在解聚态时活性较高。这些结果表明，线粒体中柠檬酸合酶的活性受到细丝形成的调节。

Recent cloning of a cDNA encoding Tetrahymena 14nm filament protein, indicated that its primary structure exhibits a high sequenceidentity with porcine citrate synthase. This led to the hypothesis that 14nm filament protein has dual functions as a citrate synthase in mitochondria and as the cytoskeleton in the cytoplasm.To examine this hypothesis, we compared antigenecity and properties of enzyme activity between purified citrate synthase and 14nm filament protein. Anti-14nm filament protein antibody cross-reacted with citrate synthase and inhibited its enzyme activity. The enzyme properties of these proteins were identical.To determine the number of the gene and mRNA of 14nm filament protein, Southern and Northern hybridization were performed. These results indicated that Tetrahymena possesses only single-type gene and a single-type mRNA of 14nm filament protein. Thus we concluded that one protein encoded from a single gene has two functions as a citrate synthase in mitochondria and as a 14nm filament protein in the cytoskeleton.Immunoelectron microscopy showed that 14nm filament protein/citrate synthase formed filament bundles in mitochondrial matrix. To clarify the relationships between filament formation and citrate synthase activity, we analyzed the change of citrate synthase activity accompanied by filament formation in vitro. Citrate synthase activity of 14nm filament protein was low in polymerized state and high in depolymerized state. These results suggested that citrate synthase activity in mitochondria was regulated by filament formation.