Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli

Summary Gram‐negative bacteria can survive in harsh environments in part because the asymmetric outer membrane (OM) hinders the entry of toxic compounds. Lipid asymmetry is established by having phospholipids (PLs) confined to the inner leaflet of the membrane and lipopolysaccharides (LPS) to the outer leaflet. Perturbation of OM lipid asymmetry, characterized by PL accumulation in the outer leaflet, disrupts proper LPS packing and increases membrane permeability. The multi‐component Mla system prevents PL accumulation in the outer leaflet of the OM via an unknown mechanism. Here, we demonstrate that in Escherichia coli, the Mla system maintains OM lipid asymmetry with the help of osmoporin OmpC. We show that the OM lipoprotein MlaA interacts specifically with OmpC and OmpF. This interaction is sufficient to localize MlaA lacking its lipid anchor to the OM. Removing OmpC, but not OmpF, causes accumulation of PLs in the outer leaflet of the OM in stationary phase, as was previously observed for MlaA. We establish that OmpC is an additional component of the Mla system; the OmpC‐MlaA complex may function to remove PLs directly from the outer leaflet to maintain OM lipid asymmetry. Our work reveals a novel function for the general diffusion channel OmpC in lipid transport. In Escherichia coli, the Mla system is proposed to maintain outer membrane lipid asymmetry by preventing phospholipid accumulation in the outer leaflet of the membrane. The lipoprotein MlaA is believed to remove phospholipids directly from the outer leaflet of the outer membrane, albeit via an unknown mechanism. We show that osmoporin OmpC plays a role in lipid asymmetry by interacting with MlaA, and likely participating in phospholipid removal from the outer leaflet of the membrane.