Structural and functional implications of phase separation of membrane protein LacY 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 little is known about LLPS of membrane proteins. 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-condensed LacY under mild hyperosmotic stress (osmotic upshift). In osmotically stressed cells, membrane-bound biomolecular condensates also reduce deformation of the cytoplasmic membrane. 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. Phase-separation of the transport protein LacY alters its membrane localization and mobility, preserves its transport activity, reduces cell membrane stress under osmotic upshift, and LacY forms mixed condensates with the enzyme β-galactosidase LacZ.