Precursor proteins are translocated across the cytoplasmic membrane in Escherichia coli by the general secretory, or Sec, pathway. The main components of the pathway are the integral membrane heterotrimeric SecYEG complex and the peripheral membrane ATPase, SecA. In this study, we have applied an in vitro assay using inverted cytoplasmic membrane vesicles to investigate the complex cycle that leads to translocation. We compared the apparent rate constants for nine precursors under two experimental conditions, single turnover and multiple turnovers. For each precursor, the rate constant for a single turnover was higher than for multiple turnovers, indicating that a different step limits the rate under the two conditions. We conclude that the rate-limiting step for a single turnover is an early step in the initial phase of transit through the channel, whereas the rate of multiple turnovers is limited by the resetting of the translocon. The presence of the chaperone SecB during multiple turnovers increased the maximal amplitude translocated for the three precursor species tested, pGBP, pPhoA, and proOmpA, and also increased the apparent rate constants for both pGBP and pPhoA. The rate constant for proOmpA was decreased by the presence of SecB.

中文翻译：

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大肠杆菌中SecYEG的单次和多次周转量的比较

前体蛋白通过一般的分泌或Sec途径跨过大肠埃希氏菌的细胞质膜。该途径的主要成分是完整的膜异源三聚体SecYEG复合物和外周膜ATPase SecA。在这项研究中，我们已经应用了体外倒置胞质膜囊泡进行化学分析以研究导致易位的复杂循环。我们比较了两种实验条件下单次翻转和多次翻转的九种前体的表观速率常数。对于每种前体，单次转换的速率常数高于多次转换的速率常数，表明在这两种条件下，不同的步骤会限制速率。我们得出的结论是，单次营业额的限速步骤是通过渠道过境的初始阶段的早期步骤，而多次营业额的比率则受到转运子的重置的限制。伴侣SecB在多次翻转过程中的存在增加了所测试的三种前体物质pGBP，pPhoA和proOmpA的最大幅度转移，并且还增加了pGBP和pPhoA的表观速率常数。SecB的存在降低了proOmpA的速率常数。