Department of Molecular Microbiology, Groningen Biomolecular Science and Biotechnology Institute, The Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
EMBO J. 2011 Sep 6;30(21):4387-97. doi: 10.1038/emboj.2011.314.
The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.
三聚体 SecYEG 复合物包含细菌细胞质膜中的蛋白质传导通道。SecYEG 与马达蛋白 SecA 一起在蛋白前体易位中发挥作用。在这里，我们研究了 SecYEG 在主动参与蛋白前体易位时的功能寡聚状态。我们将标记有荧光标记的功能性 SecYEG 复合物在原生低浓度下重构成巨大的单分子层囊泡。具有单分子灵敏度的Förster 共振能量转移和荧光（交叉）相关光谱允许独立观察 SecYEG 和前体蛋白的动力学以及复合物的形成。在 ATP 和 SecA 的存在下，多达 80%的 SecYEG 复合物与蛋白前体易位中间体结合。与 SecA 的相互作用或蛋白前体易位都不会导致 SecYEG 寡聚体的形成，而当通过交联强制形成时可以检测到这些寡聚体。这些数据表明，SecYEG 单体足以在脂质膜中形成功能性转位器。
