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 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.
Data availability
Raw microscopy, single-molecule displacement mapping, and β-Galactosidase activity assay data generated in this study are available at DataverseNL repository69: [https://doi.org/10.34894/X8GI6H]. The raw counts per minute from the transport assays are available in the Source data file. The simulation input files and trajectories are available on Zenodo70: [https://doi.org/10.5281/zenodo.17335657]. Unless otherwise stated, all data supporting the results of this study can be found in the article, supplementary, and source data files. Source data are provided with this paper.
Code availability
Code for the analysis of the molecular dynamic simulations available on Zenodo: [https://doi.org/10.5281/zenodo.17335657]. The developed code for modulating laser pulses, using a PCI-6602 programmable card (National Instruments), for SMdM analysis and PALM reconstruction is available at [https://doi.org/10.5281/zenodo.5911836] and [https://doi.org/10.5281/zenodo.14334015] as links to the Github repository of Membrane Enzymology Laboratory: https://github.com/MembraneEnzymology/].
References
Azaldegui, C. A., Vecchiarelli, A. G. & Biteen, J. S. The emergence of phase separation as an organizing principle in bacteria. Biophys. J. 120, 1123–1138 (2021).
Alberti, S. & Hyman, A. A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing. Nat. Rev. Mol. Cell Biol. 22, 196–213 (2021).
Spannl, S., Tereshchenko, M., Mastromarco, G. J., Ihn, S. J. & Lee, H. O. Biomolecular condensates in neurodegeneration and cancer. Traffic 20, 890–911 (2019).
Saini, B. & Mukherjee, T. K. Biomolecular condensates regulate enzymatic activity under a crowded milieu: synchronization of liquid–liquid phase separation and enzymatic transformation. J. Phys. Chem. B 127, 180–193 (2023).
Ginell, G. M. & Holehouse, A. S. An Introduction to the stickers-and-spacers framework as applied to biomolecular condensates. In Phase-Separated Biomolecular CondensatesVol. 2563 (eds Zhou, H.-X., Spille, J.-H. & Banerjee, P. R.) 95–116 (Springer US, 2023).
Holehouse, A. S. & Alberti, S. Molecular determinants of condensate composition. Mol. Cell 85, 290–308 (2025).
Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).
Heinkel, F. et al. Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 116, 16326–16331 (2019).
Tan, W. et al. Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub. Nat. Commun. 13, 7181 (2022).
Tusnády, G. E., Dobson, L. & Tompa, P. Disordered regions in transmembrane proteins. Biochim. Biophys. Acta (BBA)—Biomembr. 1848, 2839–2848 (2015).
Khandelwal, N. K. & Tomasiak, T. M. Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1. Nat. Commun. 15, 2389 (2024).
Fiedorczuk, K. et al. The structures of protein kinase A in complex with CFTR: mechanisms of phosphorylation and noncatalytic activation. Proc. Natl. Acad. Sci. USA 121, e2409049121 (2024).
Ukleja, M. et al. Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains. Nat. Commun. 15, 5583 (2024).
López, D. & Kolter, R. Functional microdomains in bacterial membranes. Genes Dev. 24, 1893–1902 (2010).
Gohrbandt, M. et al. Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria. EMBO J. 41, e109800 (2022).
Mangiarotti, A. & Dimova, R. Biomolecular condensates in contact with membranes. Annu. Rev. Biophys. 53, 319–341 (2024).
Lasker, K. et al. The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems. Nat. Commun. 13, 5643 (2022).
Hoang, Y. et al. An experimental framework to assess biomolecular condensates in bacteria. Nat. Commun. 15, 3222 (2024).
Bürgi, J., Xue, B., Uversky, V. N. & Van Der Goot, F. G. Intrinsic disorder in transmembrane proteins: roles in signaling and topology prediction. PLoS ONE 11, e0158594 (2016).
Xiang, L., Chen, K., Yan, R., Li, W. & Xu, K. Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity. Nat. Methods 17, 524–530 (2020).
Śmigiel, W. M. et al. Protein diffusion in Escherichia coli cytoplasm scales with the mass of the complexes and is location dependent. Sci. Adv. 8, eabo5387 (2022).
Linnik, D., Maslov, I., Punter, C. M. & Poolman, B. Dynamic structure of E. coli cytoplasm: supramolecular complexes and cell aging impact spatial distribution and mobility of proteins. Commun. Biol. 7, 508 (2024).
Yan, R., Chen, K. & Xu, K. Probing nanoscale diffusional heterogeneities in cellular membranes through multidimensional single-molecule and super-resolution microscopy. J. Am. Chem. Soc. 142, 18866–18873 (2020).
Bailey, M. W., Bisicchia, P., Warren, B. T., Sherratt, D. J. & Männik, J. Evidence for divisome localization mechanisms independent of the Min system and SlmA in Escherichia coli. PLoS Genet. 10, e1004504 (2014).
Stracy, M. et al. Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins. Mol. Cell 81, 1499–1514.e6 (2021).
Takamori, S., Cicuta, P., Takeuchi, S. & Di Michele, L. DNA-assisted selective electrofusion (DASE) of Escherichia coli and giant lipid vesicles. Nanoscale 14, 14255–14267 (2022).
Sun, Y., Sun, T.-L. & Huang, H. W. Physical properties of Escherichia coli spheroplast membranes. Biophys. J. 107, 2082–2090 (2014).
Roberts, E., Magis, A., Ortiz, J. O., Baumeister, W. & Luthey-Schulten, Z. Noise contributions in an inducible genetic switch: a whole-cell simulation study. PLoS Comput. Biol. 7, e1002010 (2011).
Culham, D. E., Romantsov, T. & Wood, J. M. Roles of K+, H+, H2O, and ΔΨ in solute transport mediated by major facilitator superfamily members ProP and LacY. Biochemistry 47, 8176–8185 (2008).
Mika, J. T., Van Den Bogaart, G., Veenhoff, L., Krasnikov, V. & Poolman, B. Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress. Mol. Microbiol. 77, 200–207 (2010).
Elowitz, M. B., Surette, M. G., Wolf, P.-E., Stock, J. B. & Leibler, S. Protein mobility in the cytoplasm of Escherichia coli. J. Bacteriol. 181, 197–203 (1999).
Harris, R., Veretnik, S., Dewan, S., Baruch Leshem, A. & Lampel, A. Regulation of enzymatic reactions by chemical composition of peptide biomolecular condensates. Commun. Chem. 7, 90 (2024).
Smirnova, I. et al. Oversized galactosides as a probe for conformational dynamics in LacY. Proc. Natl. Acad. Sci. USA 115, 4146–4151 (2018).
Renner, L. D. & Weibel, D. B. Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes. Proc. Natl. Acad. Sci. USA 108, 6264–6269 (2011).
Romantsov, T. et al. Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli: cardiolipin and osmoregulation in Escherichia coli. Mol. Microbiol. 64, 1455–1465 (2007).
Santos, T. M. A., Lin, T., Rajendran, M., Anderson, S. M. & Weibel, D. B. Polar localization of Escherichia coli chemoreceptors requires an intact Tol–Pal complex. Mol. Microbiol. 92, 985–1004 (2014).
Rafelski, S. M. & Theriot, J. A. Mechanism of polarization of Listeria monocytogenes surface protein ActA. Mol. Microbiol. 59, 1262–1279 (2006).
De Pedro, M. A., Grünfelder, C. G. & Schwarz, H. Restricted mobility of cell surface proteins in the polar regions of Escherichia coli. J. Bacteriol. 186, 2594–2602 (2004).
Lolkema, J. S. & Poolman, B. Uncoupling in secondary transport proteins. J. Biol. Chem. 270, 12670–12676 (1995).
Henderson, R. K., Fendler, K. & Poolman, B. Coupling efficiency of secondary active transporters. Curr. Opin. Biotechnol. 58, 62–71 (2019).
Mangiarotti, A. et al. Biomolecular condensates modulate membrane lipid packing and hydration. Nat. Commun. 14, 6081 (2023).
Juers, D. H., Matthews, B. W. & Huber, R. E. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 21, 1792–1807 (2012).
Bickers, S. C., Sayewich, J. S. & Kanelis, V. Intrinsically disordered regions regulate the activities of ATP binding cassette transporters. Biochim. Biophys. Acta (BBA)—Biomembr. 1862, 183202 (2020).
Kassem, N., Kassem, M. M., Pedersen, S. F., Pedersen, P. A. & Kragelund, B. B. Yeast recombinant production of intact human membrane proteins with long intrinsically disordered intracellular regions for structural studies. Biochim. Biophys. Acta (BBA)—Biomembr. 1862, 183272 (2020).
Ravindran, R. & Michnick, S. W. Biomolecular condensates as drivers of membrane trafficking and remodelling. Curr. Opin. Cell Biol. 89, 102393 (2024).
Neidhardt, F. C., Bloch, P. L. & Smith, D. F. Culture Medium for Enterobacteria. J. Bacteriol. 119, 736–747 (1974).
MembraneEnzymology. MembraneEnzymology/smdm: SMdM Analysis in Escherichia Coli Cytoplasm https://doi.org/10.5281/ZENODO.5911836 (2022).
MembraneEnzymology. MembraneEnzymology/PALM_Reconstruction: PALM Reconstruction v1.0 https://doi.org/10.5281/ZENODO.14334015 (2024).
McDonald, K. L. & Webb, R. I. Freeze substitution in 3 hours or less: freeze substiution in 3 hours or less. J. Microsc. 243, 227–233 (2011).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Abraham, M. et al. GROMACS 2024.3 Manual. https://zenodo.org/records/10589697 (2024).
Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).
Souza, P. C. T. et al. Martini 3: a general purpose force field for coarse-grained molecular dynamics. Nat. Methods 18, 382–388 (2021).
Kroon, P. et al. Martinize2 and Vermouth provide a unified framework for topology generation. eLife 12, RP90627 (2025).
Kumar, H. et al. Lactose Permease Complex with Thiodigalactoside and Nanobody 9043: 6vbg https://doi.org/10.2210/pdb6vbg/pdb (2020).
Smirnova, I., Kasho, V., Sugihara, J. & Kaback, H. R. Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding. Proc. Natl. Acad. Sci. USA 108, 15147–15151 (2011).
Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015).
Ruff, K. M. & Pappu, R. V. AlphaFold and implications for intrinsically disordered proteins. J. Mol. Biol. 433, 167208 (2021).
Wang, L., Brasnett, C., Borges-Araújo, L., Souza, P. C. T. & Marrink, S. J. Martini3-IDP: improved Martini 3 force field for disordered proteins. Nat. Commun. 16, 2874 (2025).
Westendorp, M. S. S. et al. Compartment-guided assembly of large-scale molecular models with Bentopy. Protein Science 35, e70480 (2026).
Wassenaar, T. A., Ingólfsson, H. I., Böckmann, R. A., Tieleman, D. P. & Marrink, S. J. Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations. J. Chem. Theory Comput. 11, 2144–2155 (2015).
Pogozheva, I. D. et al. Comparative molecular dynamics simulation studies of realistic eukaryotic, prokaryotic, and archaeal membranes. J. Chem. Inf. Model. 62, 1036–1051 (2022).
De Jong, D. H., Baoukina, S., Ingólfsson, H. I. & Marrink, S. J. Martini straight: boosting performance using a shorter cutoff and GPUs. Comput. Phys. Commun. 199, 1–7 (2016).
Kim, H., Fábián, B. & Hummer, G. Neighbor list artifacts in molecular dynamics simulations. J. Chem. Theory Comput. 19, 8919–8929 (2023).
Gowers, R. et al. MDAnalysis: a Python Package for the Rapid Analysis of Molecular Dynamics Simulations 98–105 (The Office of Scientific and Technical Information, 2016).
Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem. 32, 2319–2327 (2011).
Humphrey, W., Dalke, A. & Schulten, K. V. M. D. Visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).
Linnik, D. et al. Structural and Functional Implications of Phase Separation of Membrane Protein LacY in Escherichia coli (DataverseNL, 2026).
Stevens, J. A. & Marrink, S. Simulation Data: Structural and Functional Implications of Phase Separation of Membrane Protein LacY in Escherichia coli https://doi.org/10.5281/ZENODO.17335657 (2026).
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).
Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580 (1983).
Acknowledgements
We would like to thank Lyan van der Sleen for data discussions, José Vila Chã Losa and Matthias Heinemann for E. coli LY177, BW25113 ∆lacY, and Jelmer Coenradij for the purified TrxA-mEos3.2 protein. The work of Dmitrii Linnik, Jan A. Stevens, Siewert-Jan Marrink, and Bert Poolman was funded by the NWO National Science Program “The limits to growth” (grant number NWA.1292.19.170). Additionally, the work of Bert Poolman was supported by the NWO Gravitation program “Building a synthetic cell” (BaSyC). Ivan Maslov thanks the European Union for funding his research under the HORIZON TMA MSCA Postdoctoral Fellowships action (project MemProDx, 101149735).
Author information
Authors and Affiliations
Contributions
D.L. and S.S. cloned and expressed the genes. D.L., S.S., and I.M. performed wide-field fluorescence microscopy and FRAP measurements. D.L. performed SMdM and confocal measurements. D.L. performed experiments with nucleoid degradation, division inhibition, and spheroplasts formation. J.A.S. performed the MD simulations. G.K.S.-W. performed 14C-lactose uptake experiments and SDS–PAGE. R.de.B. performed high-pressure freezing and transmission electron microscopy. C.M.P. provided IT supervision and helped D.L. with analysis methods development. D.L. and B.P. conceptualized the project. D.L., I.M., J.A.S., S.J.M., and B.P. analyzed and discussed the data. Manuscript was written by D.L., I.M., and B.P. with contribution from all authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Linnik, D., Sultanji, S., Stevens, J.A. et al. Structural and functional implications of phase separation of membrane protein LacY in Escherichia coli. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69951-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-69951-7
