Durant Rhiannon J, Jarvis R Paul, Bolla Jani R
Molecular Plant Biology, Department of Biology, University of Oxford, Oxford, UK.
Commun Biol. 2025 Aug 22;8(1):1265. doi: 10.1038/s42003-025-08689-2.
The outer membranes of Gram-negative bacteria and endosymbiotic organelles mitochondria and chloroplasts contain β-barrel proteins essential for transporting metabolites, ions and proteins, as well as regulatory functions. While the assembly mechanisms of these proteins are well-studied in bacteria and mitochondria, they remain poorly understood in chloroplasts due to challenges in producing sufficient quantities of relevant plant proteins for structural and biochemical analyses. Here, we show that Oep80, the presumed core component of the chloroplastic β-barrel assembly machinery, can be overexpressed in bacteria and refolded in large quantities. While the soluble POTRA domain caused the heterologous protein to aggregate, we found that the isolated β-barrel membrane domain (Oep80) is very stable and refolds well. Using native mass spectrometry, we further show that purified Oep80 binds predicted β-signals found in several substrate β-barrel proteins in an anti-parallel manner. Overall, our findings support a central role for Oep80 in chloroplast β-barrel biogenesis. They provide evidence that the chloroplastic β-barrel assembly machinery uses a β-signal to recognise its substrates, suggesting mechanistic parallels with the BAM and SAM complexes of Gram-negative bacteria and mitochondria, respectively.
革兰氏阴性菌的外膜以及内共生细胞器线粒体和叶绿体都含有β-桶状蛋白，这些蛋白对于代谢物、离子和蛋白质的运输以及调节功能至关重要。虽然这些蛋白在细菌和线粒体中的组装机制已得到充分研究，但由于难以生产足够数量的相关植物蛋白用于结构和生化分析，它们在叶绿体中的组装机制仍知之甚少。在这里，我们表明，叶绿体β-桶状组装机制的假定核心成分Oep80可以在细菌中过表达并大量重折叠。虽然可溶性POTRA结构域导致异源蛋白聚集，但我们发现分离的β-桶状膜结构域（Oep80）非常稳定且重折叠良好。使用原生质谱，我们进一步表明纯化的Oep80以反平行方式结合几种底物β-桶状蛋白中发现的预测β信号。总体而言，我们的研究结果支持Oep80在叶绿体β-桶状生物合成中起核心作用。它们提供了证据，表明叶绿体β-桶状组装机制使用β信号来识别其底物，分别暗示了与革兰氏阴性菌和线粒体的BAM和SAM复合物在机制上的相似之处。
