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

我们研究了细胞机制中的蛋白质相互作用，这种机制介导了蛋白质在大肠杆菌细胞质膜上的转运。我们发现，在蛋白质转位酶的膜包埋的SecY-SecE-SecG复合体中，SecE和SecG各自独立地与中央提交的SecY相互作用。SecY的残基240对于SecY与SecE的相互作用尤为重要。我们建立了一个利用SecY相互作用蛋白Syd和一个削弱SecY-SecE相互作用的Secy突变的系统，在这个系统中我们可以操纵SecYEG通道复合体的SecA结合能力。SECA是前蛋白驱动的…酶，在三磷酸腺苷和前蛋白的作用下插入到膜上，并在三磷酸腺苷水解时从膜上脱出，从而促进前蛋白进入膜内。我们鉴定了一个SECY突变(SECY205)，它削弱了SECA插入反应，并推测它改变了易位通道，使改变后的通道不能接受SECA分子ATPase更多的LE与前蛋白形成复合体。我们分离了克服SecY205缺陷的SecA中的抑制突变，并对突变蛋白进行了生化鉴定。这些研究表明，SecY与SecA特异地相互作用，允许插入前蛋白-SecA复合体，而SecA插入反应对体内和体外的蛋白质转位确实是重要的。我们的突变分析表明，SecA中两个Walker A基序周围的区域对于功能上的SecA相互作用是重要的，而二级ATP结合区对于SecA功能的调节是重要的。一些SecA突变抑制了SecG的缺失突变，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。我们已经证明了SecA是通过与SecY的相互作用而被激活的，并且SecY的第五个细胞质结构域对SecA的激活是重要的。我们鉴定了SecY这个结构域中的特定残基，这对SecA的激活至关重要。较少

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