Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae

Autoři: Cole S. Sitron aff001;  Joseph H. Park aff001;  Jenna M. Giafaglione aff001;  Onn Brandman aff001
Působiště autorů: Department of Biochemistry, Stanford University, Stanford, CA, United States of America aff001;  Department of Chemical & Systems Biology, Stanford University, Stanford, CA, United States of America aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.

Klíčová slova:

Flow cytometry – Immunoblotting – Polypeptides – Ribosomes – Saccharomyces cerevisiae – Soil perturbation – Threonine – Yeast


1. Moore SD, Sauer RT. The tmRNA system for translational surveillance and ribosome rescue. Annu Rev Biochem. 2007;76: 101–124. doi: 10.1146/annurev.biochem.75.103004.142733 17291191

2. Brandman O, Hegde RS. Ribosome-associated protein quality control. Nat Struct Mol Biol. 2016;23: 7–15. doi: 10.1038/nsmb.3147 26733220

3. Defenouillère Q, Fromont-Racine M. The ribosome-bound quality control complex: from aberrant peptide clearance to proteostasis maintenance. Current Genetics. 2017. pp. 997–1005. doi: 10.1007/s00294-017-0708-5 28528489

4. Ikeuchi K, Izawa T, Inada T. Recent Progress on the Molecular Mechanism of Quality Controls Induced by Ribosome Stalling. Front Genet. 2018;9: 743. doi: 10.3389/fgene.2018.00743 30705686

5. Joazeiro CAP. Mechanisms and functions of ribosome-associated protein quality control. Nat Rev Mol Cell Biol. 2019;20: 368–383. doi: 10.1038/s41580-019-0118-2 30940912

6. Simms CL, Yan LL, Zaher HS. Ribosome Collision Is Critical for Quality Control during No-Go Decay. Mol Cell. 2017;68: 361–373.e5. doi: 10.1016/j.molcel.2017.08.019 28943311

7. Juszkiewicz S, Chandrasekaran V, Lin Z, Kraatz S, Ramakrishnan V, Hegde RS. ZNF598 Is a Quality Control Sensor of Collided Ribosomes. Mol Cell. 2018;72: 469–481.e7. doi: 10.1016/j.molcel.2018.08.037 30293783

8. Ikeuchi K, Tesina P, Matsuo Y, Sugiyama T, Cheng J, Saeki Y, et al. Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways. EMBO J. 2019;38: e100276. doi: 10.15252/embj.2018100276 30609991

9. Tsuboi T, Kuroha K, Kudo K, Makino S, Inoue E, Kashima I, et al. Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3’ end of aberrant mRNA. Mol Cell. 2012;46: 518–529. doi: 10.1016/j.molcel.2012.03.013 22503425

10. Shao S, von der Malsburg K, Hegde RS. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation. Mol Cell. 2013;50: 637–648. doi: 10.1016/j.molcel.2013.04.015 23685075

11. Sundaramoorthy E, Leonard M, Mak R, Liao J, Fulzele A, Bennett EJ. ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation. Mol Cell. 2017;65: 751–760.e4. doi: 10.1016/j.molcel.2016.12.026 28132843

12. Juszkiewicz S, Hegde RS. Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination. Mol Cell. 2017;65: 743–750.e4. doi: 10.1016/j.molcel.2016.11.039 28065601

13. Matsuo Y, Ikeuchi K, Saeki Y, Iwasaki S, Schmidt C, Udagawa T, et al. Ubiquitination of stalled ribosome triggers ribosome-associated quality control. Nat Commun. 2017;8: 159. doi: 10.1038/s41467-017-00188-1 28757607

14. Shoemaker CJ, Eyler DE, Green R. Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science. 2010;330: 369–372. doi: 10.1126/science.1192430 20947765

15. Pisareva VP, Skabkin MA, Hellen CUT, Pestova TV, Pisarev AV. Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 2011;30: 1804–1817. doi: 10.1038/emboj.2011.93 21448132

16. Lyumkis D, dos Passos DO, Tahara EB, Webb K, Bennett EJ, Vinterbo S, et al. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex. Proceedings of the National Academy of Sciences. 2014;111: 15981–15986.

17. Sitron CS, Park JH, Brandman O. Asc1, Hel2, and Slh1 couple translation arrest to nascent chain degradation. RNA. 2017;23: 798–810. doi: 10.1261/rna.060897.117 28223409

18. Bengtson MH, Joazeiro CAP. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature. 2010;467: 470–473. doi: 10.1038/nature09371 20835226

19. Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li G-W, et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell. 2012;151: 1042–1054. doi: 10.1016/j.cell.2012.10.044 23178123

20. Defenouillère Q, Yao Y, Mouaikel J, Namane A, Galopier A, Decourty L, et al. Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products. Proc Natl Acad Sci U S A. 2013;110: 5046–5051. doi: 10.1073/pnas.1221724110 23479637

21. Verma R, Oania RS, Kolawa NJ, Deshaies RJ. Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome. Elife. 2013;2: e00308. doi: 10.7554/eLife.00308 23358411

22. Abo T, Ueda K, Sunohara T, Ogawa K, Aiba H. SsrA-mediated protein tagging in the presence of miscoding drugs and its physiological role in Escherichia coli. Genes to Cells. 2002. pp. 629–638. doi: 10.1046/j.1365-2443.2002.00549.x 12081641

23. Moore SD, Sauer RT. Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli. Mol Microbiol. 2005;58: 456–466. doi: 10.1111/j.1365-2958.2005.04832.x 16194232

24. Lytvynenko I, Paternoga H, Thrun A, Balke A, Müller TA, Chiang CH, et al. Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control. Cell. 2019;178: 76–90.e22. doi: 10.1016/j.cell.2019.05.002 31155236

25. Oussenko IA, Abe T, Ujiie H, Muto A, Bechhofer DH. Participation of 3′-to-5′ exoribonucleases in the turnover of Bacillus subtilis mRNA. J Bacteriol. 2005;187: 2758–2767. doi: 10.1128/JB.187.8.2758-2767.2005 15805522

26. Choe Y-J, Park S-H, Hassemer T, Körner R, Vincenz-Donnelly L, Hayer-Hartl M, et al. Failure of RQC machinery causes protein aggregation and proteotoxic stress. Nature. 2016;531: 191–195. doi: 10.1038/nature16973 26934223

27. Kostova KK, Hickey KL, Osuna BA, Hussmann JA, Frost A, Weinberg DE, et al. CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides. Science. 2017;357: 414–417. doi: 10.1126/science.aam7787 28751611

28. Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, et al. Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A. 2006;103: 425–430. doi: 10.1073/pnas.0510013103 16407165

29. Chaudhuri RR, Allen AG, Owen PJ, Shalom G, Stone K, Harrison M, et al. Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH). BMC Genomics. 2009;10: 291. doi: 10.1186/1471-2164-10-291 19570206

30. Julio SM, Heithoff DM, Mahan MJ. ssrA (tmRNA) Plays a Role inSalmonella enterica Serovar Typhimurium Pathogenesis. J Bacteriol. 2000;182: 1558–1563. doi: 10.1128/jb.182.6.1558-1563.2000 10692360

31. Mann B, van Opijnen T, Wang J, Obert C, Wang Y-D, Carter R, et al. Control of virulence by small RNAs in Streptococcus pneumoniae. PLoS Pathog. 2012;8: e1002788. doi: 10.1371/journal.ppat.1002788 22807675

32. Chu J, Hong NA, Masuda CA, Jenkins BV, Nelms KA, Goodnow CC, et al. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration. Proc Natl Acad Sci U S A. 2009;106: 2097–2103. doi: 10.1073/pnas.0812819106 19196968

33. Ishimura R, Nagy G, Dotu I, Zhou H, -L. Yang X, Schimmel P, et al. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration. Science. 2014;345: 455–459. doi: 10.1126/science.1249749 25061210

34. Wu Z, Tantray I, Lim J, Chen S, Li Y, Davis Z, Sitron C, Dong J, Gispert S, Auburger G, Brandman O, Bi X, Snyder M, Lu B. MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure. Mol Cell. 2019;75: 835–848. doi: 10.1016/j.molcel.2019.06.031 31378462

35. Sitron CS, Brandman O. CAT tails drive degradation of stalled polypeptides on and off the ribosome. Nat Struct Mol Biol. 2019;26: 450–459. doi: 10.1038/s41594-019-0230-1 31133701

36. Shen PS, Park J, Qin Y, Li X, Parsawar K, Larson MH, et al. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science. 2015;347: 75–78. doi: 10.1126/science.1259724 25554787

37. Osuna BA, Howard CJ, Kc S, Frost A, Weinberg DE. In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing. Elife. 2017; e27949. doi: 10.7554/eLife.27949 28718767

38. Yonashiro R, Tahara EB, Bengtson MH, Khokhrina M, Lorenz H, Chen K-C, et al. The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation. Elife. 2016;5: e11794. doi: 10.7554/eLife.11794 26943317

39. Defenouillère Q, Zhang E, Namane A, Mouaikel J, Jacquier A, Fromont-Racine M. Rqc1 and Ltn1 prevent CAT-tail induced protein aggregation by efficient recruitment of Cdc48 on stalled 60S subunits. J Biol Chem. 2016;291: 12245–12253. doi: 10.1074/jbc.M116.722264 27129255

40. Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N. Computer Control of Microscopes Using μManager. Current Protocols in Molecular Biology. 2010. doi: 10.1002/0471142727.mb1420s92 20890901

41. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Pagé N, et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 2001;294: 2364–2368. doi: 10.1126/science.1065810 11743205

42. Letzring DP, Dean KM, Grayhack EJ. Control of translation efficiency in yeast by codon–anticodon interactions. RNA. 2010;16: 2516–2528. doi: 10.1261/rna.2411710 20971810

43. Eaglestone SS, Ruddock LW, Cox BS, Tuite MF. Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2000;97: 240–244. doi: 10.1073/pnas.97.1.240 10618402

44. Ness F, Ferreira P, Cox BS, Tuite MF. Guanidine Hydrochloride Inhibits the Generation of Prion “Seeds” but Not Prion Protein Aggregation in Yeast. Mol Cell Biol. 2002;22: 5593–5605. doi: 10.1128/MCB.22.15.5593-5605.2002 12101251

45. Byrne LJ, Cox BS, Cole DJ, Ridout MS, Morgan BJT, Tuite MF. Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride. Proc Natl Acad Sci U S A. 2007;104: 11688–11693. doi: 10.1073/pnas.0701392104 17606924

46. Ferreira PC, Ness F, Edwards SR, Cox BS, Tuite MF. The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation. Mol Microbiol. 2001;40: 1357–1369. doi: 10.1046/j.1365-2958.2001.02478.x 11442834

47. Jung G, Masison DC. Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions. Curr Microbiol. 2001;43: 7–10. doi: 10.1007/s002840010251 11375656

48. Jung G, Jones G, Masison DC. Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance. Proc Natl Acad Sci U S A. 2002;99: 9936–9941. doi: 10.1073/pnas.152333299 12105276

49. Grimminger V, Richter K, Imhof A, Buchner J, Walter S. The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104. J Biol Chem. 2004;279: 7378–7383. doi: 10.1074/jbc.M312403200 14668331

50. Donnelly MLL, Ryan MD, Mehrotra A, Gani D, Hughes LE, Luke G, et al. Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal “skip.” Journal of General Virology. 2001. pp. 1013–1025. doi: 10.1099/0022-1317-82-5-1013 11297676

51. Szymczak AL, Vignali DAA. Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther. 2005;5: 627–638. doi: 10.1517/14712598.5.5.627 15934839

52. Åkerfelt M, Trouillet D, Mezger V, Sistonen LEA. Heat shock factors at a crossroad between stress and development. Ann N Y Acad Sci. 2007;1113: 15–27. doi: 10.1196/annals.1391.005 17483205

53. Morimoto RI. The heat shock response: systems biology of proteotoxic stress in aging and disease. Cold Spring Harb Symp Quant Biol. 2011;76: 91–99. doi: 10.1101/sqb.2012.76.010637 22371371

54. Sorger PK, Pelham HR. Purification and characterization of a heat-shock element binding protein from yeast. EMBO J. 1987;6: 3035–3041. 3319580

55. Mirón-García MC, Garrido-Godino AI, García-Molinero V, Hernández-Torres F, Rodríguez-Navarro S, Navarro F. The prefoldin bud27 mediates the assembly of the eukaryotic RNA polymerases in an rpb5-dependent manner. PLoS Genet. 2013;9: e1003297. doi: 10.1371/journal.pgen.1003297 23459708

56. Yan Z, Costanzo M, Heisler LE, Paw J, Kaper F, Andrews BJ, et al. Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers. Nature Methods. 2008. pp. 719–725. doi: 10.1038/nmeth.1231 18622398

57. Turowski TW, Tollervey D. Transcription by RNA polymerase III: insights into mechanism and regulation. Biochem Soc Trans. 2016;44: 1367–1375. doi: 10.1042/BST20160062 27911719

58. Deplazes A, Möckli N, Luke B, Auerbach D, Peter M. Yeast Uri1p promotes translation initiation and may provide a link to cotranslational quality control. EMBO J. 2009;28: 1429–1441. doi: 10.1038/emboj.2009.98 19387492

59. Breslow DK, Cameron DM, Collins SR, Schuldiner M, Stewart-Ornstein J, Newman HW, et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods. 2008;5: 711–718. doi: 10.1038/nmeth.1234 18622397

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden