Multifunction of Tetrahymena citrate synthase

最近对四膜虫14nm丝蛋白cDNA的克隆表明，其一级结构与猪柠檬酸合酶具有较高的序列一致性。这导致了14nm丝蛋白在线粒体中具有柠檬酸合成酶和细胞质中具有细胞骨架的双重功能的假设。为了验证这一假设，我们比较了纯化的柠檬酸合酶和14nm丝蛋白的抗原性和酶活性特性。抗14nm丝蛋白抗体与柠檬酸合酶发生交叉反应，抑制其酶活性。这些蛋白质的酶性质是相同的。采用南、北杂交法测定14nm丝蛋白基因数量和mRNA表达量。这些结果表明，四膜虫只具有单一型基因和14nm丝蛋白的单一型mRNA。因此，我们得出结论，一个由单一基因编码的蛋白在线粒体中具有柠檬酸合成酶和在细胞骨架中具有14nm丝蛋白的两种功能。免疫电镜显示，14nm丝蛋白/柠檬酸合成酶在线粒体基质中形成丝束。为了阐明成丝与柠檬酸合酶活性之间的关系，我们在体外分析了柠檬酸合酶活性随成丝的变化。14nm丝状蛋白的柠檬酸合酶活性在聚合状态下低，在解聚合状态下高。这些结果表明，线粒体中的柠檬酸合成酶活性受细丝形成的调控。

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.