Phase separation of a yeast prion protein promotes cellular fitness
Biophysical responses of proteins to stress
Much recent work has focused on liquid-liquid phase separation as a cellular response to changing physicochemical conditions. Because phase separation responds critically to small changes in conditions such as pH, temperature, or salt, it is in principle an ideal way for a cell to measure and respond to changes in the environment. Small pH changes could, for instance, induce phase separation of compartments that store, protect, or inactivate proteins. Franzmann et al. used the yeast translation termination factor Sup35 as a model for a phase separation–induced stress response. Lowering the pH induced liquid-liquid phase separation of Sup35. The resulting liquid compartments subsequently hardened into gels, which sequestered the termination factor. Raising the pH triggered dissolution of the gels, concomitant with translation restart. Protecting Sup35 in gels could provide a fitness advantage to recovering yeast cells that must restart the translation machinery after stress.
Science, this issue p. eaao5654
Structured Abstract
INTRODUCTION
The formation of dynamic, membraneless compartments using intracellular phase transitions such as phase separation and gelation provides an efficient way for cells to respond to environmental changes. Recent work has identified a special class of intrinsically disordered domains enriched for polar amino acids such as glycine, glutamine, serine, or tyrosine as potential drivers of phase separation in cells. However, more traditional work has highlighted the ability of these domains to drive the formation of fibrillar aggregates. Such domains are also known as prion domains. They have first been identified in budding yeast proteins that form amyloid-like aggregates. Because these aggregates are heritable and change the activity of the prion-domain–containing protein, they are thought to be a common mechanism for phenotypic inheritance in fungi and other organisms. However, the aggregation of prion domains has also been associated with neurodegenerative diseases in mammals. Therefore, the relationship between the role of these domains as drivers of phase separation and their ability to form prion-like aggregates is unknown.
RATIONALE
The budding yeast translation termination factor Sup35 is an archetypal prion-domain–containing protein. Sup35 forms irreversible heritable aggregates, and these aggregates have been proposed to be either a disease or an adaptation that generates heritable phenotypic variation in populations of budding yeast. Despite having been described almost 25 years ago, the physiological functions of the Sup35 prion domain and other prion-like domains remain unclear. Uncovering these functions is a prerequisite for understanding the evolutionary pressures shaping prion-like sequences and how their physiological and pathological transitions affect cellular fitness.
RESULTS
Here, we show that the prion domain of Sup35 drives the reversible phase separation of the translation termination factor into biomolecular condensates. These condensates are distinct and different from fibrillar amyloid-like prion particles. Combining genetic analysis in cells with in vitro reconstitution protein biochemistry and quantitative biophysical methods, we demonstrate that Sup35 condensates form by pH-induced liquid-like phase separation as a response to sudden stress. The condensates are liquid-like initially but subsequently solidify to form protective protein gels. Cryo–electron tomography demonstrates that these gel-like condensates consist of cross-linked Sup35 molecules forming a porous meshwork. A cluster of negatively charged amino acids functions as a pH sensor and regulates condensate formation. The ability to form biomolecular condensates is shared among distantly related budding yeast and fission yeast. This suggests that condensate formation is a conserved and ancestral function of the prion domain of Sup35. In agreement with an important physiological function of the prion domain, the catalytic guanosine triphosphatase (GTPase) domain of the translation termination factor Sup35 readily forms irreversible aggregates in the absence of the prion domain. Consequently, cells lacking the prion domain exhibit impaired translational activity and a growth defect when recovering from stress. These data demonstrate that the prion domain rescues the essential GTPase domain of Sup35 from irreversible aggregation, thus ensuring that the translation termination factor remains functional during harsh environmental conditions.
CONCLUSION
The prion domain of Sup35 is a highly regulated molecular device that has the ability to sense and respond to physiochemical changes within cells. The N-terminal prion domain provides the interactions that drive liquid phase separation. Phase separation is regulated by the adjacent stress sensor. The synergy of these two modules enables the essential translation termination factor to rapidly form protective condensates during stress. This suggests that prion domains are protein-specific stress sensors and modifiers of protein phase transitions that allow cells to respond to specific environmental conditions.
The Sup35 prion domain regulates phase separation of the translation termination factor Sup35 during cellular stress.
The translation termination factor Sup35 (depicted in the magnifying glass) consists of a disordered prion domain (cyan) a disordered stress sensor domain (red) and a folded catalytic domain (blue). During growth, Sup35 catalyzes translation termination. During cell stress, the prion domain and the sensor domain act together to promote phase separation into protective and reversible biomolecular condensates.
Abstract
Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.
Get full access to this article
View all available purchase options and get full access to this article.
Supplementary Material
Summary
Figs. S1 to S5
Tables S1 and S2
Movies S1 to S8
Resources
References and Notes
1
S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Biomolecular condensates: Organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
2
C. P. Brangwynne, C. R. Eckmann, D. S. Courson, A. Rybarska, C. Hoege, J. Gharakhani, F. Jülicher, A. A. Hyman, Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
3
A. Patel, H. O. Lee, L. Jawerth, S. Maharana, M. Jahnel, M. Y. Hein, S. Stoynov, J. Mahamid, S. Saha, T. M. Franzmann, A. Pozniakovski, I. Poser, N. Maghelli, L. A. Royer, M. Weigert, E. W. Myers, S. Grill, D. Drechsel, A. A. Hyman, S. Alberti, A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).
4
A. Molliex, J. Temirov, J. Lee, M. Coughlin, A. P. Kanagaraj, H. J. Kim, T. Mittag, J. P. Taylor, Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).
5
J. A. Riback, C. D. Katanski, J. L. Kear-Scott, E. V. Pilipenko, A. E. Rojek, T. R. Sosnick, D. A. Drummond, Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168, 1028–1040 (2017).
6
M. Kato, T. W. Han, S. Xie, K. Shi, X. Du, L. C. Wu, H. Mirzaei, E. J. Goldsmith, J. Longgood, J. Pei, N. V. Grishin, D. E. Frantz, J. W. Schneider, S. Chen, L. Li, M. R. Sawaya, D. Eisenberg, R. Tycko, S. L. McKnight, Cell-free formation of RNA granules: Low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).
7
D. M. Garcia, D. F. Jarosz, Rebels with a cause: Molecular features and physiological consequences of yeast prions. FEMS Yeast Res. 14, 136–147 (2014).
8
R. Halfmann, S. Lindquist, Epigenetics in the extreme: Prions and the inheritance of environmentally acquired traits. Science 330, 629–632 (2010).
9
R. Halfmann, S. Alberti, S. Lindquist, Prions, protein homeostasis, and phenotypic diversity. Trends Cell Biol. 20, 125–133 (2010).
10
A. F. Harrison, J. Shorter, RNA-binding proteins with prion-like domains in health and disease. Biochem. J. 474, 1417–1438 (2017).
11
R. B. Wickner, [URE3] as an altered URE2 protein: Evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566–569 (1994).
12
P. Anderson, N. Kedersha, Stress granules. Curr. Biol. 19, R397–R398 (2009).
13
J. R. Buchan, R. Parker, Eukaryotic stress granules: The ins and outs of translation. Mol. Cell 36, 932–941 (2009).
14
J. Shorter, S. Lindquist, Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers. Science 304, 1793–1797 (2004).
15
Y. Shin, C. P. Brangwynne, Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
16
M. C. Munder, D. Midtvedt, T. Franzmann, E. Nüske, O. Otto, M. Herbig, E. Ulbricht, P. Müller, A. Taubenberger, S. Maharana, L. Malinovska, D. Richter, J. Guck, V. Zaburdaev, S. Alberti, A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. eLife 5, 59–69 (2016).
17
I. Petrovska, E. Nüske, M. C. Munder, G. Kulasegaran, L. Malinovska, S. Kroschwald, D. Richter, K. Fahmy, K. Gibson, J. M. Verbavatz, S. Alberti, Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation. eLife 2014, (2014).
18
B. R. Parry, I. V. Surovtsev, M. T. Cabeen, C. S. O’Hern, E. R. Dufresne, C. Jacobs-Wagner, The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity. Cell 156, 183–194 (2014).
19
S. Saha, C. A. Weber, M. Nousch, O. Adame-Arana, C. Hoege, M. Y. Hein, E. Osborne-Nishimura, J. Mahamid, M. Jahnel, L. Jawerth, A. Pozniakovski, C. R. Eckmann, F. Jülicher, A. A. Hyman, Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism. Cell 166, 1572–1584 (2016).
20
T. R. Serio, A. G. Cashikar, A. S. Kowal, G. J. Sawicki, J. J. Moslehi, L. Serpell, M. F. Arnsdorf, S. L. Lindquist, Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289, 1317–1321 (2000).
21
S. Alberti, R. Halfmann, O. King, A. Kapila, S. Lindquist, A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137, 146–158 (2009).
22
I. Stansfield, K. M. Jones, V. V. Kushnirov, A. R. Dagkesamanskaya, A. I. Poznyakovski, S. V. Paushkin, C. R. Nierras, B. S. Cox, M. D. Ter-Avanesyan, M. F. Tuite, The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 14, 4365–4373 (1995).
23
M. D. Ter-Avanesyan, V. V. Kushnirov, A. R. Dagkesamanskaya, S. A. Didichenko, Y. O. Chernoff, S. G. Inge-Vechtomov, V. N. Smirnov, Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein. Mol. Microbiol. 7, 683–692 (1993).
24
Y. O. Chernoff, A. P. Galkin, E. Lewitin, T. A. Chernova, G. P. Newnam, S. M. Belenkiy, Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein. Mol. Microbiol. 35, 865–876 (2000).
25
H. K. Edskes, H. J. Khamar, C.-L. Winchester, A. J. Greenler, A. Zhou, R. P. McGlinchey, A. Gorkovskiy, R. B. Wickner, Sporadic distribution of prion-forming ability of Sup35p from yeasts and fungi. Genetics 198, 605–616 (2014).
26
T. K. Harris, G. J. Turner, Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53, 85–98 (2002).
27
F. Ruggeri, F. Zosel, N. Mutter, M. Różycka, M. Wojtas, A. Ożyhar, B. Schuler, M. Krishnan, Single-molecule electrometry. Nat. Nanotechnol. 12, 488–495 (2017).
28
J. R. Glover, A. S. Kowal, E. C. Schirmer, M. M. Patino, J.-J. Liu, S. Lindquist, Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell 89, 811–819 (1997).
29
M. Rubinstein, A. N. Semenov, Thermoreversible gelation in solutions of associating polymers. 2. Linear dynamics. Macromolecules 31, 1386–1397 (1998).
30
T. S. Harmon, A. S. Holehouse, M. K. Rosen, R. V. Pappu, Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. eLife 6, e30294 (2017).
31
M.-T. Wei, S. Elbaum-Garfinkle, A. S. Holehouse, C. C.-H. Chen, M. Feric, C. B. Arnold, R. D. Priestley, R. V. Pappu, C. P. Brangwynne, Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat. Chem. 9, 1118–1125 (2017).
32
L. Z. Osherovich, J. S. Weissman, Multiple Gln/Asn-rich prion domains confer susceptibility to induction of the yeast [PSI+] prion. Cell 106, 183–194 (2001).
33
S. V. Paushkin, V. V. Kushnirov, V. N. Smirnov, M. D. Ter-Avanesyan, Propagation of the yeast prion-like [psi+] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor. EMBO J. 15, 3127–3134 (1996).
34
R. P. McGlinchey, D. Kryndushkin, R. B. Wickner, Suicidal [PSI+] is a lethal yeast prion. Proc. Natl. Acad. Sci. U.S.A. 108, 5337–5341 (2011).
35
R. B. Wickner, D. C. Masison, H. K. Edskes, [PSI] and [URE3] as yeast prions. Yeast 11, 1671–1685 (1995).
36
T. Nakayashiki, C. P. Kurtzman, H. K. Edskes, R. B. Wickner, Yeast prions [URE3] and [PSI+] are diseases. Proc. Natl. Acad. Sci. U.S.A. 102, 10575–10580 (2005).
37
R. B. Wickner, H. K. Edskes, D. Bateman, A. C. Kelly, A. Gorkovskiy, The yeast prions [PSI+] and [URE3] are molecular degenerative diseases. Prion 5, 258–262 (2011).
38
Y. O. Chernoff, Stress and prions: Lessons from the yeast model. FEBS Lett. 581, 3695–3701 (2007).
39
L. Malinovska, S. Alberti, Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem. Prion 9, 339–346 (2015).
40
L. Malinovska, S. Palm, K. Gibson, J.-M. Verbavatz, S. Alberti, Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation. Proc. Natl. Acad. Sci. U.S.A. 112, E2620–E2629 (2015).
41
S. Alberti, A. D. Gitler, S. Lindquist, A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913–919 (2007).
42
J. Bähler, J.-Q. Wu, M. S. Longtine, N. G. Shah, A. McKenzie 3rd, A. B. Steever, A. Wach, P. Philippsen, J. R. Pringle, Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).
43
S. L. Forsburg, N. Rhind, Basic methods for fission yeast. Yeast 23, 173–183 (2006).
44
R. Serrano, Energy requirements for maltose transport in yeast. Eur. J. Biochem. 80, 97–102 (1977).
45
M. J. Mahon, pHluorin2: An enhanced, ratiometric, pH-sensitive green florescent protein. Adv. Biosci. Biotechnol. 2, 132–137 (2011).
46
C. L. Brett, D. N. Tukaye, S. Mukherjee, R. Rao, The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking. Mol. Biol. Cell 16, 1396–1405 (2005).
47
R. Halfmann, S. Lindquist, Screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis. J. Vis. Exp. 17, e838 (2008).
48
T. Mašek, L. Valášek, M. Pospíšek, in RNA: Methods in Molecular Biology (Methods and Protocols), vol. 703, H. Nielsen, Ed. (Humana Press, 2011), pp. 293–309.
49
X. Li, P. Mooney, S. Zheng, C. R. Booth, M. B. Braunfeld, S. Gubbens, D. A. Agard, Y. Cheng, Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).
50
D. N. Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
51
M. Jahnel, M. Behrndt, A. Jannasch, E. Schäffer, S. W. Grill, Measuring the complete force field of an optical trap. Opt. Lett. 36, 1260–1262 (2011).
52
A. S. Holehouse, R. K. Das, J. N. Ahad, M. O. G. Richardson, R. V. Pappu, CIDER: Resources to analyze sequence-ensemble relationships of intrinsically disordered proteins. Biophys. J. 112, 16–21 (2017).
53
Z. Dosztányi, V. Csizmok, P. Tompa, I. Simon, IUPred: Web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).
54
J. Gough, K. Karplus, R. Hughey, C. Chothia, Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J. Mol. Biol. 313, 903–919 (2001).
(0)eLetters
eLetters is an online forum for ongoing peer review. Submission of eLetters are open to all. eLetters are not edited, proofread, or indexed. Please read our Terms of Service before submitting your own eLetter.
Log In to Submit a ResponseNo eLetters have been published for this article yet.
Information & Authors
Information
Published In
Science
Volume 359 | Issue 6371
5 January 2018
5 January 2018
Copyright
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
This is an article distributed under the terms of the Science Journals Default License.
Submission history
Received: 3 August 2017
Accepted: 27 November 2017
Published in print: 5 January 2018
Acknowledgments
We thank the following Services and Facilities of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) for their support: We thank B. Borgonovo and R. Lemaitre (Protein Expression Purification and Characterization) for help with protein expression and purification; B. Nitzsche and B. Schroth-Diez (Light Microscopy Facility) for help with light microscopy; F. Friedrich for support with visualization (Media Technologies and Outreach); and C. Iserman for a yeast strain BY4742 coexpressing Sup35-GFP and Pab1-mCherry. We thank members of the MPI-CBG and C. Weber from the Max Planck Institute for the Physics of Complex systems for discussion and comments on the manuscript. R. Halfmann is acknowledged for providing the antibody against Sup35C domain. We gratefully acknowledge funding from the German Federal Ministry of Research and Education (BMBF 031A359A to T.M.F and A.A.H.). This work is supported by the MaxSynBio consortium, jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society. We further acknowledge the U.S. National Institutes of Health for grant 5RO1NS056114 to R.V.P., The Human Frontiers Program for grant RGP0034/2017 to S.A. and R.V.P, the Volkswagen “Life?” initiative for a grant to S.A., and the German Research Foundation (DFG) for a grant to S.A. All the data relevant to this study are included in the main paper or the supplementary materials.
Authors
Funding Information
National Institutes of Health: 5RO1NS056114
BMBF MaxSynBio: 031A359A
Metrics & Citations
Metrics
Article Usage
Altmetrics
Citations
Export citation
Select the format you want to export the citation of this publication.
Cited by
- Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation, Science Advances, 8, 13, (2022)./doi/10.1126/sciadv.abm5149
- Polymerization in the actin ATPase clan regulates hexokinase activity in yeast, Science, 367, 6481, (1039-1042), (2021)./doi/10.1126/science.aay5359
- Valence and patterning of aromatic residues determine the phase behavior of prion-like domains, Science, 367, 6478, (694-699), (2021)./doi/10.1126/science.aaw8653
- Protein condensates as aging Maxwell fluids, Science, 370, 6522, (1317-1323), (2021)./doi/10.1126/science.aaw4951
- The glassiness of hardening protein droplets, Science, 370, 6522, (1271-1272), (2021)./doi/10.1126/science.abe9745
- A unique route of colloidal phase separation yields stress-free gels, Science Advances, 6, 41, (2020)./doi/10.1126/sciadv.abb8107
- Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets, Science Advances, 6, 23, (2020)./doi/10.1126/sciadv.aba3471
- Exploiting mammalian low-complexity domains for liquid-liquid phase separation–driven underwater adhesive coatings, Science Advances, 5, 8, (2019)./doi/10.1126/sciadv.aax3155
Loading...
View Options
Check Access
Log in to view the full text
AAAS login provides access to Science for AAAS Members, and access to other journals in the Science family to users who have purchased individual subscriptions.
- Become a AAAS Member
- Activate your AAAS ID
- Purchase Access to Other Journals in the Science Family
- Account Help
Log in via OpenAthens.
Log in via Shibboleth.
More options
Register for free to read this article
As a service to the community, this article is available for free. Login or register for free to read this article.
Buy a single issue of Science for just $15 USD.