Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanistic dissection of increased enzymatic rate in a phase-separated compartment

Nature Chemical Biology volume 17, pages 693–702 (2021)Cite this article

Subjects

Abstract

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid–liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Design of an inducible, condensate-targeted enzyme cascade.
Fig. 2: Condensates increase total SUMOylation rate.
Fig. 3: Rate enhancement is substrate dependent.
Fig. 4: SUMOylation is greatly accelerated in the droplet phase.
Fig. 5: Excess activity is due to a scaffold-induced decrease in KM.
Fig. 6: Activity enhancement is scaffold specific.

Data availability

All raw data have been deposited in the Dryad Database: https://datadryad.org/stash/share/ZBLh5JFV-KjDQjCmqiX1qSFQbWJ3CXsd9Eu-l8rpxkM. Source data are provided with this paper. All reagents are available upon request from the authors.

Code availability

The Matlab code used to generate Extended Data Figs. 1, 2 and 3 is provided in the Supplementary Information.

References

  1. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).

  3. Sheth, U. & Parker, R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sheu-Gruttadauria, J. & MacRae, I. J. Phase transitions in the assembly and function of human miRISC. Cell 173, 946–957 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352, 595–599 (2016).

  6. Ditlev, J. A. et al. A composition-dependent molecular clutch between T cell signaling condensates and actin. eLife https://doi.org/10.7554/eLife.42695 (2019).

  7. Lafontaine, D. L. J., Riback, J. A., Bascetin, R. & Brangwynne, C. P. The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol. Cell Biol. 22, 165–182 (2021).

    Article  CAS  PubMed  Google Scholar 

  8. Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xing, W., Muhlrad, D., Parker, R. & Rosen, M. K. A quantitative inventory of yeast P body proteins reveals principles of composition and specificity. eLife https://doi.org/10.7554/eLife.56525 (2020).

  10. Guillen-Boixet, J. et al. RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell 181, 346–361 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yang, P. et al. G3BP1 is a tunable switch that triggers phase separation to assemble stress granules. Cell 181, 325–345 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sanders, D. W. et al. Competing protein-RNA interaction networks control multiphase intracellular organization. Cell 181, 306–324 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lyon, A. S., Peeples, W. B. & Rosen, M. K. A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 22, 215–235 (2021).

    Article  CAS  PubMed  Google Scholar 

  15. Banjade, S. & Rosen, M. K. Phase transitions of multivalent proteins can promote clustering of membrane receptors. eLife 3, e04123 (2014).

    Article  PubMed Central  Google Scholar 

  16. Case, L. B., Zhang, X., Ditlev, J. A. & Rosen, M. K. Stoichiometry controls activity of phase-separated clusters of actin signaling proteins. Science 363, 1093–1097 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Woodruff, J. B. et al. The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin. Cell 169, 1066–1077 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. King, M. R. & Petry, S. Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation. Nat. Commun. 11, 270 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dewey, D. C., Strulson, C. A., Cacace, D. N., Bevilacqua, P. C. & Keating, C. D. Bioreactor droplets from liposome-stabilized all-aqueous emulsions. Nat. Commun. 5, 4670 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).

    Article  CAS  PubMed  Google Scholar 

  21. Freeman Rosenzweig, E. S. et al. The eukaryotic CO2-concentrating organelle is liquid-like and exhibits dynamic reorganization. Cell 171, 148–162 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Huang, W. Y. C. et al. A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS. Science 363, 1098–1103 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Poudyal, R. R. et al. Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates. Nat. Commun. 10, 490 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Love, C. et al. Reversible pH-responsive coacervate formation in lipid vesicles activates dormant enzymatic reactions. Angew. Chem. 59, 5950–5957 (2020).

    Article  CAS  Google Scholar 

  25. Kojima, T. & Takayama, S. Membraneless compartmentalization facilitates enzymatic cascade reactions and reduces substrate inhibition. ACS Appl. Mater. Interfaces 10, 32782–32791 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Castellana, M. et al. Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat. Biotechnol. 32, 1011–1018 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Langdon, E. M. et al. mRNA structure determines specificity of a polyQ-driven phase separation. Science 360, 922–927 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Brady, J. P. et al. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc. Natl Acad. Sci. USA 114, E8194–E8203 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Stroberg, W. & Schnell, S. Do cellular condensates accelerate biochemical reactions? Lessons from microdroplet chemistry. Biophys. J. 115, 3–8 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Schlatter, S., Senn, C. & Fussenegger, M. Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins. Biotechnol. Bioeng. 83, 210–225 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Muller, S., Hoege, C., Pyrowolakis, G. & Jentsch, S. SUMO, ubiquitin’s mysterious cousin. Nat. Rev. Mol. Cell Biol. 2, 202–210 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Varejao, N., Lascorz, J., Li, Y. & Reverter, D. Molecular mechanisms in SUMO conjugation. Biochem. Soc. Trans. 48, 123–135 (2020).

    Article  CAS  PubMed  Google Scholar 

  34. Lallemand-Breitenbach, V. & de The, H. PML nuclear bodies: from architecture to function. Curr. Opin. Cell Biol. 52, 154–161 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. Melchior, F. SUMO-nonclassical ubiquitin. Annu. Rev. Cell Dev. Biol. 16, 591–626 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Bernier-Villamor, V., Sampson, D. A., Matunis, M. J. & Lima, C. D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108, 345–356 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Coey, C. T. et al. E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex. J. Biol. Chem. 289, 15810–15819 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Balcells, C. et al. Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase. J. Phys. Chem. B 118, 4062–4068 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Park, S. H., Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Gao, Y., Roberts, C. C., Toop, A., Chang, C. E. & Wheeldon, I. Mechanisms of enhanced catalysis in enzyme-DNA nanostructures revealed through molecular simulations and experimental analysis. ChemBioChem 17, 1430–1436 (2016).

    Article  CAS  PubMed  Google Scholar 

  41. Nguyen, A. W. & Daugherty, P. S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Ditlev, J. A., Case, L. B. & Rosen, M. K. Who’s in and who’s out—compositional control of biomolecular condensates. J. Mol. Biol. 430, 4666–4684 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Küffner, A. M. et al. Acceleration of an enzymatic reaction in liquid phase separated compartments based on intrinsically disordered protein domains. ChemSystemsChem https://doi.org/10.1002/syst.202000001 (2020).

  44. Drobot, B. et al. Compartmentalised RNA catalysis in membrane-free coacervate protocells. Nat. Commun. 9, 3643 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Bouchard, J. J. et al. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments. Mol. Cell 72, 19–36 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Correll, C. C., Bartek, J. & Dundr, M. The nucleolus: a multiphase condensate balancing ribosome synthesis and translational capacity in health, aging and ribosomopathies. Cells 8, 869 (2019).

  47. Woodruff, J. B. et al. Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro. Science 348, 808–812 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yamada, K. et al. Characterization of the C-terminal diglycine motif of SUMO-1/3. Biosci. Biotechnol. Biochem. 76, 1035–1037 (2012).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Banani and A. Rice for constructs, and all members of the Rosen Laboratory, past and present, for helpful advice and discussions. Research was supported by the Howard Hughes Medical Institute, a Paul G. Allen Frontiers Group Distinguished Investigator Award and a grant from the Welch Foundation (no. I-1544, to M.K.R.).

Author information

Authors and Affiliations

  1. Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    William Peeples & Michael K. Rosen

Authors
  1. William Peeples
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael K. Rosen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.K.R. and W.P. conceived the study and designed the research program. W.P. performed all experiments. M.K.R. secured funding and supervised the work. W.P. and M.K.R. wrote the manuscript.

Corresponding author

Correspondence to Michael K. Rosen.

Ethics declarations

Competing interests

M.K.R. is a founder of Faze Medicines.

Additional information

Peer review information Nature Chemical Biology thanks Tanja Mittag and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Sensitivity of enhanced condensate activity to KM, substrate concentration, and partition coefficient.

a, Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold (TotalS and TotalUS, respectively, as a function of substrate concentration (plotted as [S]/KM,US) and partition coefficient (PC). Modeled for KM,US = 150 and KM,S = 50, as measured for FRB-polySH33 + polyPRM5, with identical PC values for enzyme and substrate. Modeling assumes simple, hyperbolic Michaelis-Menten kinetics (see Methods). Color scale is a relative representation of the z-axis values and goes from low (blue) to high (red). Inset is a plot of TotalS:TotalUS rate as a function of substrate concentration at a fixed partition coefficient of 50. b, Modeled ratio of TotalS to TotalUS as a function of PC and the change in KM upon recruitment of enzyme and substrate to the scaffold, KM,US/KM,S. Total substrate concentration, [S]T, set to 0.1 * KM,US. c, Same as (b), except [S]T set to 10 * KM,US.

Extended Data Fig. 2 Total scaffold rate can be less than total unscaffolded activity in certain regimes if enzyme partitioning is much less than substrate partitioning.

a-c, Modeled ratio of total reaction rate in a phase separated solution, with and without recruitment of enzyme and substrate to the scaffold as a function of substrate concentration and substrate partition coefficient (PCS). Both reactions have KM = 150. Enzyme partitioning (PCE) is 1 (a), 10 (b), and 100 (c); enzyme concentration, [E] = 0.1[S]. Model based on 0.01 droplet volume fraction.

Extended Data Fig. 3 Droplet rate increases rapidly relative to bulk as a function of partition coefficient.

a, Modeled ratio of droplet and bulk reaction rates as a function of substrate concentration and partition coefficient (PC). Both reactions are scaffolded and have KM = 50. Enzyme partitioning is identical to substrate partitioning, and [E] = 0.1[S]. b, Modeled fractional activity contributed by the droplet phase as a function of substrate concentration and partition coefficient (PC). Conditions same as in (a), with a 0.01 droplet volume fraction.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Tables 1 and 2 and Method (modeling).

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed gels for Fig. 2b,d,f.

Source Data Fig. 3

Unprocessed gels for Fig. 3a,c,

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Peeples, W., Rosen, M.K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. Nat Chem Biol 17, 693–702 (2021). https://doi.org/10.1038/s41589-021-00801-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00801-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing