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<p>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 <i>Escherichia coli</i>, 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<sup>Pop</sup>. Molecular dynamics simulations show how the PopTag domain drives the condensate-like association dynamics of LacY<sup>Pop</sup> through hydrophobic sticker interactions. LacY<sup>Pop</sup> 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<sup>Pop</sup> at the poles of <i>E. coli</i>. 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月25日

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  • Dmitrii Linnik,
  • Sumayra Sultanji,
  • Jan A. Stevens,
  • Gea K. Schuurman-Wolters,
  • Rinse de Boer,
  • Christiaan M. Punter,
  • Siewert J. Marrink,
  • Ivan Maslov,
  • Bert Poolman

摘要

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.