Multifunction of Tetrahymena citrate synthase

最近克隆了一个编码四膜虫14 nm细丝蛋白的cDNA，表明其一级结构与猪柠檬酸合成酶具有较高的序列同源性。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的性质。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。为确定14 nm细丝蛋白的基因和信使核糖核酸的数目，进行了Southern和Northern杂交。这些结果表明，四膜虫只具有单一类型的基因和14 nm细丝蛋白的单一类型的mRNA.因此，我们认为一个基因编码的蛋白质在线粒体中具有柠檬酸合成酶和细胞骨架中14 nm细丝蛋白的双重功能。免疫电子显微镜显示14 nm细丝蛋白/柠檬酸合成酶在线粒体基质中形成了细丝束。为了阐明微丝形成与柠檬酸合成酶活性之间的关系，我们在体外分析了微丝形成过程中柠檬酸合成酶活性的变化。14 nm细丝蛋白的柠檬酸合成酶活性在聚合态较低，解聚态较高。这些结果表明线粒体中的柠檬酸合成酶活性受微丝形成的调节。

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.