双精氨酸易位(Tat)系统将折叠的蛋白质转运穿过细菌质膜和叶绿体类囊体膜。这里,我们研究了来自大肠杆菌的三种不同纯化的Tat复合物的组成和结构组织,鼠伤寒沙门氏菌和根癌农杆菌。首先,我们证明了这些Tat系统在体内的功能活性,由于tatABC操纵子在大肠杆菌tatnull突变株中从鼠伤寒S.typhimurium或A.tumefaciens的表达导致了含有大肠杆菌辅因子的底物的有效Tat依赖性输出,TMAO还原酶。三个孤立的,亲和标记的Tat复合物包含TatA,TatB和TatC在每种情况下,证明这三个亚基之间有很强的相互作用。所有三种复合物的单粒子电子显微镜研究显示近似椭圆形,最大尺寸为13nm的不对称颗粒。一个共同的特征是围绕或多或少的中央污点池的许多污点排除密度,提示有蛋白质衬里的孔或洞。颗粒之间尺寸变化的特征表明组装的模块化形式和/或不同数量的TatBC/TatA单元的募集。尽管序列同源性较低,组合数据表明这三种细菌的Tat系统中的结构和功能保守性。
The twin-arginine translocation (Tat) system transports folded proteins across bacterial plasma membranes and the chloroplast thylakoid membrane. Here, we investigate the composition and structural organization of three different purified Tat complexes from Escherichia coli, Salmonella typhimurium and Agrobacterium tumefaciens. First, we demonstrate the functional activity of these Tat systems in vivo, since expression of the tatABC operons from S.typhimurium or A.tumefaciens in an E.coli tat null mutant strain resulted in efficient Tat-dependent export of an E.coli cofactor-containing substrate, TMAO reductase. The three isolated, affinity-tagged Tat complexes comprised TatA, TatB and TatC in each case, demonstrating a strong interaction between these three subunits. Single-particle electron microscopy studies of all three complexes revealed approximately oval-shaped, asymmetric particles with maximal dimensions up to 13 nm. A common feature is a number of stain-excluding densities surrounding more or less central pools of stain, suggesting protein-lined pores or cavities. The characteristics of size variation among the particles suggest a modular form of assembly and/or the recruitment of varying numbers of TatBC/TatA units. Despite low levels of sequence homology, the combined data indicate structural and functional conservation in the Tat systems of these three bacterial species.