Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
J Mol Biol. 2013 Nov 15;425(22):4145-53. doi: 10.1016/j.jmb.2013.08.012. Epub 2013 Aug 20.
Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation.
最近的研究提出了一种新的观点，即 SecYEG 转运通道作为单体发挥作用，并与二聚体 SecA ATP 酶相互作用，为前体蛋白易位反应提供动力。在这里，我们使用纳米盘重建的 SecYEG 来表征转运通道的单拷贝功能特性。我们首次使用基于分子间Förster 共振能量转移的方法表明，分离的纳米盘重建的 SecYEG 单体支持前体蛋白易位。当几个 SecYEG 拷贝共同重建在纳米盘中时，没有观察到易位动力学的变化，这表明 SecYEG 寡聚体不会促进增强的易位。相比之下，纳米盘重建的 SecYEG PrlA4 变体单体显示出增加的易位速率。基于纳米盘分离的单体 SecYEG 内分子间Förster 共振能量转移的实验表明，与 SecA 相互作用后，通道会发生核苷酸依赖性开放。总之，纳米盘重建的 SecYEG 单体可用于前体蛋白易位，为蛋白易位动态方面的单分子分析提供了新的前景。
