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

Abstract

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 LacY^Pop. Molecular dynamics simulations show how the PopTag domain drives the condensate-like association dynamics of LacY^Pop through hydrophobic sticker interactions. LacY^Pop 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 LacY^Pop 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.