Hirst T R, Holmgren J
Department of Genetics, University of Leicester, Great Britain.
Proc Natl Acad Sci U S A. 1987 Nov;84(21):7418-22. doi: 10.1073/pnas.84.21.7418.
The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, we show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [35S]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of approximately 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of approximately 13 min, we conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be approximately 20 and approximately 260 micrograms/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. Our findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.
霍乱弧菌肠毒素的分泌过程中，毒素亚基会短暂进入周质间隙。在本文中，我们发现这些亚基在通过外膜分泌之前，会在周质间隙内寡聚形成完整毒素。通过用[35S] - 甲硫氨酸脉冲标记细胞，然后监测放射性标记亚基在周质间隙内组装时的周转情况，研究了毒素组装的速率。亚基以单体形式进入周质间隙，并在大约1分钟的半衰期内组装成寡聚体。由于与毒素从周质间隙流出的速率相比，组装是一个快速事件，毒素流出的半衰期约为13分钟，因此我们得出结论，所有穿过周质间隙的亚基在穿过外膜之前就已组装完成。计算得出周质间隙内亚基单体和组装好的完整毒素的平均浓度分别约为20微克/毫升和约260微克/毫升。这表明周质间隙是一个浓度适宜的环境，能够发生毒素的自发组装。我们的研究结果表明，与通过其他生物膜的输出明显不同，蛋白质穿过细菌外膜涉及已经折叠成三级甚至四级构象的多肽的转运。
