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细胞质膜易位的细胞机制中的蛋白质相互作用。我们发现，在蛋白转位酶的膜包埋的SecY-SecE-SecG复合体中，SecE和SecG各自独立地与中心递质SecY相互作用。SecY的残数240对于SecY与SecE的相互作用尤为重要。我们建立了一个系统，使用一个secY相互作用蛋白Syd和一个破坏secY - sece相互作用的secY突变，我们可以操纵SecYEG通道复合物的seca结合能力。SecA是一种驱动前蛋白的ATP酶，它在ATP和前蛋白的作用下插入膜中，并在ATP水解时从膜中脱插入，从而促进前蛋白向膜中的移动。我们发现了一种破坏SecA插入反应的secY突变（SecY205），并认为它改变了易位通道，使改变的通道不能接受与前蛋白复杂的SecA分子。我们在SecA中分离出克服SecY205缺陷的抑制突变，并对突变蛋白进行了生化表征。这些研究表明，SecY与SecA特异性相互作用，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易位确实很重要。我们的突变体分析表明，SecA中两个Walker A基序周围的区域对SecA功能相互作用很重要，而次级atp结合区域对SecA功能的调节很重要。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进行蛋白质易位的作用。我们已经证明SecA通过与SecY的相互作用而被激活，并且SecY的第五个细胞质结构域对SecA的激活很重要。我们在SecA的这个结构域中发现了特殊的残基，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