Schesser K, Frithz-Lindsten E, Wolf-Watz H
Department of Cell and Molecular Biology, University of Umea, Sweden.
J Bacteriol. 1996 Dec;178(24):7227-33. doi: 10.1128/jb.178.24.7227-7233.1996.
Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and, furthermore, that YerA is not required to target YopE-Cya proteins to the secretion complex. Taken together, our results suggest that traversing the bacterial and eukaryotic membranes occurs by separate processes that recognize distinct domains of YopE and that these processes are not dependent on YerA activity.
致病性耶尔森氏菌通过Ⅲ型分泌系统将多种不同的效应分子（称为Yops）递送至真核细胞的胞质溶胶中。为了确定假结核耶尔森氏菌中YopE的哪些区域对于其穿过细菌和真核细胞膜是必需的，我们构建了一系列杂交基因，这些基因由不同长度的yopE与百日咳博德特氏菌环腺苷酸合成酶基因（cyaA）的腺苷酸环化酶编码结构域融合而成。通过检测完整细胞的腺苷酸环化酶活性，我们发现仅包含YopE的前11个氨基末端残基的YopE-Cya蛋白能有效地输出到细菌细胞的外表面。YopE前七个残基的单个氨基酸替换显著降低了在细胞表面检测到的报告蛋白的量，这表明YopE的极端氨基末端区域被分泌机制识别。正如最近对小肠结肠炎耶尔森氏菌YopE蛋白所显示的那样（M.-P. Sory、A. Boland、I. Lambermont和G. R. Cornelis，《美国国家科学院院刊》92:11998 - 12002，1995），我们发现YopE-Cya蛋白输出到细胞表面并不足以使其递送至真核细胞质中。为了穿过HeLa细胞膜，至少需要49个yopE编码的残基。将YopE的亮氨酸43替换为甘氨酸严重影响了报告蛋白递送至HeLa细胞。令人惊讶的是，从细菌细胞输出也不足以使YopE-Cya蛋白从细菌细胞表面释放到培养上清液中。在培养上清液中检测相应报告蛋白的活性至少需要75个YopE残基，这表明YopE的该区域存在一个释放结构域。我们还表明，伴侣样蛋白YerA在体外与YopE-Cya蛋白形成稳定复合物至少需要75个YopE残基，此外，将YopE-Cya蛋白靶向分泌复合物并不需要YerA。综上所述，我们的结果表明，穿过细菌膜和真核膜是通过识别YopE不同结构域的不同过程发生的，并且这些过程不依赖于YerA的活性。
