SecY functions that support the dynamic movement of SecA, a protein-translocating ATPase

我们研究了介导蛋白质跨大肠杆菌易位的细胞机制中的蛋白质相互作用。coli细胞质膜。我们发现，在膜包埋的SecY-SecE-SecG复合物的蛋白质移位酶，每个SecE和SecG相互作用独立的SecY，中央提交。SecY的残基240对于SecY与SecE的相互作用特别重要。我们建立了一个系统，使用SecY相互作用蛋白质，Syd，和一个secY突变，损害SecY-SecE相互作用，在其中我们可以操纵SecYEG通道复合物的SecA结合能力。SecA是前蛋白驱动的ATP酶，在ATP和前蛋白的作用下插入细胞膜，ATP水解后从细胞膜上脱嵌，从而促进前蛋白进入细胞膜。我们发现了一个secY突变（SecY 205），它损害了SecA插入反应，并认为它改变了转运通道，使改变的通道不能接受SecA分子。 ...更多信息 即与前蛋白复合。我们在SecA中分离出克服SecY 205缺陷的抑制突变，并对突变蛋白进行生物化学表征。这些研究表明，SecY特异性地与SecA相互作用以允许前蛋白-SecA复合物的插入，并且SecA插入反应对于体内和体外的蛋白质移位确实是重要的。我们的突变体分析表明，SecA中的两个步行者A基序周围的区域是重要的功能SecA的相互作用，而第二ATP结合区是重要的SecA功能的调节。一些SecA突变抑制了SecG缺失突变，我们的分析表明SecG具有辅助SecA及其有效利用ATP进行蛋白质转运的作用。我们已经表明，SecA是通过其与SecY的相互作用而激活的，并且SecY的第五胞质结构域对于SecA的激活是重要的。我们对SecY的这个结构域中的特定残基进行了修饰，这对SecA的激活至关重要。少

We studied protein interactions in the cellular machinery that mediates protein translocation across the E. coli cytoplasmic membrane. We found that in the membrane-embedded SecY-SecE-SecG complex of protein translocase, each of SecE and SecG interacts independently with SecY, the central submit. The residue 240 of SecY is particularly important for the SecY's interaction with SecE. We established a system, using a SecY-interaction protein, Syd, and a secY mutation that impairs SecY-SecE interaction, in which we can manipulate the SecA-binding ability of the SecYEG channel complex. SecA, the preprotein-driving ATPase, inserts into the membrane in response to ATP and a preprotein and deinserts from the membrane upon hydrolysis of ATP, thereby facilitating the preprotein movement into the membrane, We identified a secY mutation(SecY205)that impairs the SecA insertion reaction, and suggested that it alters the translocation channel such that the altered channel cannot accept a SecA molecu … More le that is in complex with the preprotein. We isolated suppressor mutations in SecA that overcame the SecY205 defect, and characterized the mutant proteins biochemically. These studies suggested that SecY interacts specifically with SecA to allow the insertion of preprotein-SecA complex, and the SecA insertion reactions is indeed important for protein translocation both in vivo and in vitro. Our mutant analyses suggest that the regions around the two Walker A motifs in SecA are important for the functional SecA interaction, while the secondary ATP-binding region is important for the regulation of the SecA functions. Some SecA mutations suppressed the SecG deletion mutation and our analyses suggest that SecG has a role of assisting SecA and its effective utilization of ATP for protein translocation. We have shown that SecA is activated by its interaction with SecY, and that the fifth cytoplasmic domain of SecY is important for the SecA activation. We identhified particular residues in this domain of SecY, which is crucial for the SecA activation. Less