The SecYEG interactome of E. coli: non-competitive binding of PpiD and YidC to the SecYEG translocon

The SecYEG translocon constitutes the major protein transporting channel in bacteria and transports an enormous variety of different secretory and inner membrane proteins. The minimal core of the SecYEG translocon consists of the three inner membrane proteins SecY, SecE and SecG, which together with appropriate targeting factors are sufficient for protein transport in vitro. However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, which likely allow the SecYEG translocon to manage diverse substrates. For obtaining a global view on SecYEG plasticity, a label free proteomics approach was executed, which identified known SecYEG interacting proteins, verified the interaction with quality control proteins and identified several novel putative SecYEG interactors. Surprisingly, the chaperone complex PpiD/YfgM was identified as the most pronounced contact partner of SecYEG and was much dominant than the established partner protein YidC. Detailed analyses of the PpiD and YidC contact sites on SecY by site directed cross-linking revealed that PpiD and YidC use almost completely overlapping binding sites on SecY and contact the lateral gate, the plug domain and the periplasmic cavity of SecY. Still, despite having almost identical binding sites, their binding to SecY is non-competitive as determined by quantitative mass spectrometry and cross-linking. This implies that the SecYEG translocon forms different substrate-independent sub-assemblies, in which SecYEG is either in contact with YidC or with the PpiD/YfgM complex.

SecYEG易位通道（SecYEG translocon）是细菌体内主要的蛋白质转运通道，负责转运种类繁多的分泌蛋白与内膜蛋白。SecYEG易位通道的最小核心组分为SecY、SecE与SecG三种内膜蛋白，搭配合适的靶向因子后即可在体外实现蛋白质转运功能。但体内研究证实，SecYEG易位通道可与多种伴侣蛋白结合，这或许使其能够处理多样化的底物蛋白。为全面解析SecYEG的可塑性，研究人员采用无标记蛋白质组学（label free proteomics）方法，不仅鉴定出已知的SecYEG互作蛋白，验证了其与质控蛋白的相互作用，还发现了多个新的潜在SecYEG互作因子。令人意外的是，伴侣蛋白复合物PpiD/YfgM被鉴定为SecYEG最显著的接触伙伴，其丰度远高于已证实的互作蛋白YidC。通过定点交联（site-directed cross-linking）技术对SecY上的PpiD与YidC结合位点进行详细分析，研究人员发现二者在SecY上的结合位点几乎完全重叠，均靶向SecY的侧向门、塞结构域与周质腔。尽管二者的结合位点几乎一致，但定量质谱（quantitative mass spectrometry）与交联实验结果显示，它们与SecY的结合并不存在竞争关系。这表明SecYEG易位通道可形成不同的非底物依赖型亚组装体，其中SecYEG要么与YidC结合，要么与PpiD/YfgM复合物结合。