Lee V T, Anderson D M, Schneewind O
Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles 90095, USA.
Mol Microbiol. 1998 May;28(3):593-601. doi: 10.1046/j.1365-2958.1998.00822.x.
Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1-100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.
致病性耶尔森氏菌通过将其一些Yop蛋白靶向真核细胞质来黏附并杀死巨噬细胞。关于YopE的靶向过程是作为多肽在细胞间的直接转运，还是分两个不同步骤进行，存在争议，每个步骤都需要特定信号来实现YopE穿过细菌包膜的分泌以及转运到真核细胞质中。在这里，我们使用洋地黄皂苷对真核质膜进行选择性溶解，以测量耶尔森氏菌感染HeLa细胞期间的Yop靶向。在真核细胞质中发现了YopE、YopH、YopM和YopN，但在细胞外培养基中未发现。当YopE在耶尔森氏菌细胞质中与SycE伴侣结合时，其1-100位残基对于杂合新霉素磷酸转移酶的靶向是必要且充分的。电子显微镜分析未能检测到YopE靶向的细胞外中间体，提示存在一步转运机制。
