Kirschbaum Carla, Bennett Jack L, Tian Qiaoyu, Sen Navoneel, Smith Iain P S, Wu Di, Benesch Justin L P, Khalid Syma, Isom Georgia, Robinson Carol V
Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford OX1 3QU, United Kingdom.
Department of Chemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Proc Natl Acad Sci U S A. 2025 Apr 8;122(14):e2420041122. doi: 10.1073/pnas.2420041122. Epub 2025 Apr 1.
The maintenance of lipid asymmetry (Mla) system in gram-negative bacteria transfers phospholipids between the outer and inner membrane to maintain the outer membrane asymmetry. Misplaced phospholipids are extracted from the outer leaflet of the outer membrane by MlaA, transferred to the periplasmic lipid transporter MlaC, and shuttled to the inner membrane. We set out to investigate the lipid transfer between MlaA and MlaC using native mass spectrometry, with the aim of determining the lipid preferences of MlaC and whether MlaA preselected lipids for MlaC. First, we characterized the lipids that copurified with overexpressed MlaC, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), and following delipidation noted a headgroup-independent enrichment of cyclopropane lipids. Under native expression conditions, we found that PG is three-fold enriched on MlaC compared to its abundance in the membrane. Next, we isolated and characterized OmpF-MlaA complexes and demonstrated their ability to enhance loading of delipidated MlaC with bacterial and nonbacterial phospholipids. We then captured the intact ternary lipid shuttle (OmpF-MlaA-MlaC) and demonstrated that PG dissociates this transient complex, releasing lipid-bound MlaC. Together our results point to a high population of endogenous PG on periplasmic MlaC, which likely arises from disassembly of the lipid shuttle to maintain lipid asymmetry for cell viability.
革兰氏阴性菌中的脂质不对称性维持（Mla）系统可在外膜和内膜之间转运磷脂，以维持外膜的不对称性。错位的磷脂由MlaA从外膜的外层小叶中提取出来，转移到周质脂质转运蛋白MlaC，再转运到内膜。我们着手利用原生质体质谱研究MlaA和MlaC之间的脂质转移，目的是确定MlaC的脂质偏好以及MlaA是否为MlaC预先选择脂质。首先，我们对与过表达的MlaC共纯化的脂质、磷脂酰甘油（PG）和磷脂酰乙醇胺（PE）进行了表征，并在脱脂后注意到环丙烷脂质的头基非依赖性富集。在天然表达条件下，我们发现与膜中的丰度相比，PG在MlaC上的富集程度高出三倍。接下来，我们分离并表征了OmpF-MlaA复合物，并证明了它们增强脱脂MlaC与细菌和非细菌磷脂结合的能力。然后，我们捕获了完整的三元脂质穿梭体（OmpF-MlaA-MlaC），并证明PG会使这种瞬时复合物解离，释放出与脂质结合的MlaC。我们的研究结果共同表明，周质MlaC上存在大量内源性PG，这可能是脂质穿梭体分解以维持细胞活力的脂质不对称性所导致的。
