Department of Microbiology, University of Chicago, Chicago, Illinois, USA.
Howard Taylor Ricketts Laboratory, University of Chicago, Lemont, Illinois, USA.
J Biol Chem. 2022 Sep;298(9):102318. doi: 10.1016/j.jbc.2022.102318. Epub 2022 Jul 31.
Secretion systems utilize ATPase activity to facilitate the translocation of proteins into and across membranes. In bacteria, the universally conserved SecA ATPase binds a large repertoire of preproteins and interacts with the SecYEG translocon. In contrast, the type 7b secretion system (T7bSS) of Staphylococcus aureus supports the secretion of a restricted subset of proteins. T7bSSs are found in several Firmicutes as gene clusters encoding secreted WXG100 proteins and FtsK/SpoIIIE-like ATPase. In S. aureus, this ATPase is called EssC and comprises two cytosolic forkhead-associated domains (FHA), two membrane-spanning segments (TM), and four cytosolic modules named DUF (domain of unknown function) and ATPases (D1D2D3). However, a detailed understanding of the interactions of EssC in the T7bSS is not clear. Here, we tagged EssC and performed affinity chromatography of detergent-solubilized extracts of wild type and isogenic mutants of S. aureus. We found that EssC recruits EsaA, EssA, and EssB in a complex referred to as the ESS (ESAT-6 like secretion system) translocon, and secreted substrates were not required for translocon assembly. Furthermore, deletions of FHA and DUF rendered EssC unstable, whereas FHA was required for association with EssB. This interaction was independent of EsaA, but EsaA was required to recruit EssA to the EssC-EssB complex. Finally, we show that assembly of the ESS translocon was impaired upon mutation of D2 structural motifs. Together, our data indicate that the ESS translocon is maintained fully assembled at the plasma membrane and that D2 is fundamental in sustaining the integrity of this complex.
分泌系统利用 ATP 酶活性促进蛋白质跨膜转运。在细菌中，普遍保守的 SecA ATP 酶结合了大量的前蛋白，并与 SecYEG 转运体相互作用。相比之下，金黄色葡萄球菌的 7b 型分泌系统（T7bSS）支持一小部分蛋白质的分泌。T7bSS 存在于几种厚壁菌门中，是编码分泌性 WXG100 蛋白和 FtsK/SpoIIIE 样 ATP 酶的基因簇。在金黄色葡萄球菌中，这种 ATP 酶称为 EssC，由两个胞质叉头相关结构域（FHA）、两个跨膜片段（TM）和四个胞质模块组成，分别命名为 DUF（功能未知结构域）和 ATPases（D1D2D3）。然而，对于 EssC 在 T7bSS 中的相互作用的详细了解尚不清楚。在这里，我们标记了 EssC，并对野生型和金黄色葡萄球菌同源突变体的去污剂溶解提取物进行了亲和层析。我们发现 EssC 招募 EsaA、EssA 和 EssB 形成一个复合物，称为 ESS（ESAT-6 样分泌系统）转运体，并且分泌底物不是转运体组装所必需的。此外，FHA 和 DUF 的缺失使 EssC 不稳定，而 FHA 对于与 EssB 的结合是必需的。这种相互作用不依赖于 EsaA，但 EsaA 是将 EssA 招募到 EssC-EssB 复合物所必需的。最后，我们表明 D2 结构基序的突变会损害 ESS 转运体的组装。总之，我们的数据表明 ESS 转运体在质膜上完全组装，并且 D2 对于维持该复合物的完整性是至关重要的。
