Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China.
Sci Rep. 2017 Mar 9;7:44023. doi: 10.1038/srep44023.
Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.
细菌中非经典的蛋白质分泌是一种常见现象。然而，非经典分泌途径的选择原则尚不清楚。在这里，我们的实验数据在我们所知的范围内首次表明，折叠的多聚体蛋白质可以被枯草芽孢杆菌中的非经典分泌途径识别和分泌。我们探索了来自反刍新月单胞菌 5_1_39BFAA 的典型细胞质蛋白 D-阿洛酮糖 3-差向异构酶（RDPE）的分泌模式，并表明其非经典分泌不仅仅是由于细胞裂解。截断变体的分析表明，C 端和 N 端以及两个疏水区对于 RDPE 的结构稳定性和非经典分泌是必需的。RDPE 的疏水区的丙氨酸扫描突变表明，疏水性残基介导了其折叠和未折叠形式之间的平衡。带有 RDPE 区域的报告 mCherry 和 GFP 融合显示其分泌需要完整的四聚体蛋白复合物。使用交联的四聚体，我们表明折叠的四聚体 RDPE 可以作为单个单位进行分泌。最后，我们提供的证据表明，非经典分泌途径强烈偏爱多聚体底物，这些底物在细胞的两极和隔膜区域积累。总的来说，这些数据表明多聚体识别机制可能适用于整个非经典分泌途径。
