Dimeric SecA is essential for protein translocation
Dimeric SecA is essential for protein translocation
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DOI：
10.1073/pnas.0502774102
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发表时间：
2005-05-24
影响因子：
11.1
通讯作者：
Oliver, D
中科院分区：
文献类型：
--
作者：
Jilaveanu, LB;Zito, CR;Oliver, D
SecA通过与分泌前或膜蛋白和SecYEG易位通道的结合促进细菌蛋白易位。一旦组装，SecA atp酶在SecYEG上经历膜插入和缩回的循环，以逐步的方式驱动蛋白质易位。SecA存在于单体和二聚体之间的平衡状态，与它的易位配体的结合极大地改变了这种平衡。在这里，我们研究了蛋白质易位可以通过SecA单体发生的提议。我们产生了一个缺乏残基2-11的SecA蛋白突变体，该突变体主要以单体形式存在，不能补充条件致死性SecA突变体，对体外蛋白易位不活跃，对易位atp酶活性不活跃。此外，我们开发了一种称为膜捕获的技术，其中野生型SecA亚基通过过量生产膜粘附突变SecA蛋白而被捕获在膜内，并且在一个案例中，证明了膜相关的SecA异源二聚体。最后，我们检测了内源性和重组膜结合的SecA，并发现两种情况下的SecA二聚体水平都很高，通过化学交联进行了评估。总的来说，我们的研究结果强烈表明，膜结合的SecA二聚体对蛋白质易位周期至关重要，尽管这些结果不能排除SecA单体在易位过程的某些阶段的参与。我们的发现对SecA运动功能和易位组装和激活具有重要意义。
SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal secA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.