Structural and functional implications of in vivo phase separation of membrane protein in Escherichia coli

Liquid-liquid phase-separation (LLPS) controls protein activity and dynamically organizes (macro)molecules in living systems without the need for membrane-bound compartments. Biomolecular condensates of water-soluble proteins have extensively been studied, but LLPS of membrane proteins is unchartered territory. In this work we induce in vivo condensation of lactose permease (LacY), a widely-studied model monomeric inner membrane protein in Escherichia coli, and evaluate how it affects LacY function. We fused LacY with engineered, condensate-forming protein PopTag. We observe major changes in the localization and mobility of LacYPop. Molecular dynamics simulations show how the PopTag domain drives the condensate-like association dynamics of LacYPop through hydrophobic sticker interactions. LacYPop preserves native-level transport activity and outperforms the non-condensated LacY under mild hyperosmotic stress. Perturbation experiments suggest that membrane curvature drives the accumulation of LacYPop at the poles of E. coli. Co-condensation of LacY and β-galactosidase LacZ slightly reduces their activity and results in remarkable cellular reorganization of the proteins. Our research shows the localization, dynamics, and function of phase-separated membrane proteins in bacteria and highlights the potential of LLPS for engineering complex metabolic networks in vivo.