Study on the two-component pathway composed of a stress protein and an outer membrane protein

为了确定共生素是蚜虫内共生体内两组分途径的一种感受器激酶，我寻找了可能的反应调节器，能够从共生素中获得高能的磷酰基。经SDS-PAGE分离，固定在PVDF膜上，与共生素-32&gt；P孵育后，发现8种多肽具有放射性，表明磷酸基已从共生素转移到这些多肽上。由于这8种蛋白可能在蚜虫内共生体中起反应调节作用，我们开始了对这些蛋白的研究。氨基酸序列测定和免疫化学分析表明，66、63、42和20 kDa的蛋白分别是B.aphidicola DnaK、B.aphidicola共生素、E.coliOmpF和P.milliaris组蛋白h1的同源蛋白。电子显微镜免疫组织化学分析和亚细胞分级研究表明，OmpF同源42-kDa蛋白主要定位于外膜部分，而共生素仅定位于内共生体的细胞质部分。研究还表明，共生素与分离的外膜之间的磷转移是以时间依赖的方式进行的。基于这些发现，我提出了一个由应激蛋白“共生素”和外膜蛋白“OmpF”组成的双组分途径介导的信号转导系统的模型。

In an effort to identify symbionin as a sensor kinase of the two-component pathway in the aphid endosymbint, I searched for possible response regulators that are capable of assepting the high-energy phosphoryl group from symbionin. When the endosymbiotic proteins were separated by SDS-PAGE,fixed on a PVDF membrane, and then incubated with symbionin-^<32>P,the radioactivety was found on eight species of polypeptides, indicating that the phosphoryl group was transferred from symbionin to these polypeptides. Since it is possible that these eight proteins function as response regulators in the aphid endosymbiont, we started the study on the characterization of these proteins.Amino acid sequincing and immunochemical analysis of 66,63,42, and 20-kDa proteins revealed that are homologues of B.aphidicola DnaK,B.aphidicola symbionin, E.coli OmpF,and P.milliaris Histone Hl, respectively. Immunohistochemical analysis using electron microscope and subcellular fractionation study demonstrated that OmpF-homologous 42-kDa protein is localized mainly in the outer membrane fraction, while symbionin is only in the cytoplasmic fraction of endosymbiont. It was also shown that the phosphotransfer between symbionin and isolated outer membrane proceeds in a time-dependent fashion. Based on these findings, I peoposed a model for the signal transduction system mediated by the two-component pathway consisting of a stress protein "symbionin", and an outer membrane protein "OmpF".