School of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
Living Systems Institute, University of Exeter, Exeter, U.K.
Biochem J. 2023 Feb 27;480(4):283-296. doi: 10.1042/BCJ20220480.
Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC - aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
革兰氏阴性菌被两层富含蛋白质的膜包围，其间夹有一层肽聚糖层。它们共同构成了包膜（或细胞壁），这对于能量产生、脂质生物合成、结构完整性以及抵御物理和化学环境挑战至关重要。为了实现包膜生物发生，周质和外膜蛋白（OMPs）必须从细胞质穿过内膜，通过普遍存在的 SecYEG 蛋白通道进行运输。新生蛋白要么在周质中折叠，要么穿过肽聚糖（PG）层朝向外膜，通过β桶组装机制（BAM）进行插入。由于蛋白质密度高且缺乏能量（ATP 或化学渗透势能），疏水性蛋白在周质中的运输尤其危险。许多分子伴侣有助于防止聚集和从聚集中恢复，其中 SurA 已知与 BAM 相互作用，促进其递送至外膜。然而，目前尚不清楚从 Sec 机制中出现的蛋白质在与 SurA 相互作用之前是如何被接收和保护免受聚集和蛋白水解的。通过生化分析和电子显微镜，我们证明了空载和底物结合的 SurA 与 SecYEG-SecDF-YidC 的内膜转运机制复合物（即全转运体（HTL））的结合能力。在 AlphaFold 预测的支持下，我们提出了 SecDF 的周质结构域在将伴侣招募到 SecYEG 中的蛋白转运出口位点中的作用。我们认为这种与已征募伴侣的直接相互作用有助于防止新生包膜蛋白的聚集和降解，促进它们安全地进入周质和外膜。
