CoChat
Sign Up
Cited by 89Ubiquitin and proteasome pathwaysEvolution and Genetic DynamicsProtein Structure and Dynamics

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting

Open access
Mavor, David, Barlow, Kyle, Thompson, Samuel, Barad, Benjamin A, Bonny, Alain R, Cario, Clinton L, Gaskins, Garrett, Liu, Zairan +33 more·2016·eLife
DOI 10.7554/elife.15802PMC PMC4862753

Published version of a preprint

Open this paper in CoChat — free account

Abstract

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.7554/elife.15802

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • By amplifying and sequencing barcodes from each bin and using a predetermined barcode-variant index, a functional score can be computed for each variant.

    “By amplifying barcodes and sequencing them from each bin, then using the predetermined barcode-variant index to determine the distribution of each variant across bins, a functional score can be computed for each variant (e.g. (59, 92, 93)).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [93]

    The Results (Library construction) describe a barcode-to-allele lookup table used to count alleles by sequencing barcodes. Materials and methods (EMPIRIC-BC) describes calculating fitness scores from barcode counts, but the samples are competition time points, not bins; the claimed distribution across bins is not supported by this paper. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 51

  1. Balch WE Morimoto RI Dillin A Kelly JW 2008 Adapting proteostasis for disease intervention Science 319 916 919 10.1126/science.1141448 18276881 · DOI · Paper page
  2. Benatuil L Perez JM Belk J Hsieh CM 2010 An improved yeast transformation method for the generation of very large human antibody libraries Protein Engineering, Design & Selection 23 155 159 10.1093/protein/gzq002 20130105 · DOI
  3. Boucher JI Cote P Flynn J Jiang L Laban A Mishra P Roscoe BP Bolon DN 2014 Viewing protein fitness landscapes through a next-gen lens Genetics 198 461 471 10.1534/genetics.114.168351 25316787 PMC4196605 · DOI
  4. Erpapazoglou Z Walker O Haguenauer-Tsapis R 2014 Versatile roles of k63-linked ubiquitin chains in trafficking Cells 3 3 1027 1088 10.3390/cells3041027 25396681 PMC4276913 · DOI
  5. Finley D Ozkaynak E Varshavsky A 1987 The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses Cell 48 1035 1046 10.1016/0092-8674(87)90711-2 3030556 · DOI
  6. Finley D Sadis S Monia BP Boucher P Ecker DJ Crooke ST Chau V 1994 Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant Molecular and Cellular Biology 14 5501 5509 10.1128/MCB.14.8.5501 8035826 PMC359070 · DOI
  7. Finley D Ulrich HD Sommer T Kaiser P 2012 The ubiquitin-proteasome system of Saccharomyces cerevisiae Genetics 192 319 360 10.1534/genetics.112.140467 23028185 PMC3454868 · DOI
  8. Fowler DM Stephany JJ Fields S 2014 Measuring the activity of protein variants on a large scale using deep mutational scanning Nature Protocols 9 2267 2284 10.1038/nprot.2014.153 25167058 PMC4412028 · DOI
  9. Frand AR Kaiser CA 1998 The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum Molecular Cell 1 1 161 170 10.1016/S1097-2765(00)80017-9 9659913 · DOI
  10. Fujiwara K Tenno T Sugasawa K Jee JG Ohki I Kojima C Tochio H Hiroaki H Hanaoka F Shirakawa M 2004 Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B The Journal of Biological Chemistry 279 4760 4767 10.1074/jbc.M309448200 14585839 · DOI
  11. Gardner RG Nelson ZW Gottschling DE 2005 Degradation-mediated protein quality control in the nucleus Cell 120 803 815 10.1016/j.cell.2005.01.016 15797381 · DOI
  12. Gietz RD Woods RA 2002 Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method Methods in Enzymology 350 350 87 96 10.1016/S0076-6879(02)50957-5 12073338 · DOI
  13. Hanna J Leggett DS Finley D 2003 Ubiquitin depletion as a key mediator of toxicity by translational inhibitors Molecular and Cellular Biology 23 9251 9261 10.1128/MCB.23.24.9251-9261.2003 14645527 PMC309641 · DOI
  14. Hanna J Meides A Zhang DP Finley D 2007 A ubiquitin stress response induces altered proteasome composition Cell 129 747 759 10.1016/j.cell.2007.03.042 17512408 · DOI
  15. Hietpas R Roscoe B Jiang L Bolon DN 2012 Fitness analyses of all possible point mutations for regions of genes in yeast Nature Protocols 7 1382 1396 10.1038/nprot.2012.069 22722372 PMC3509169 · DOI
  16. Hietpas RT Bank C Jensen JD Bolon DN 2013 Shifting fitness landscapes in response to altered environments Evolution 67 3512 3522 10.1111/evo.12207 24299404 PMC3855258 · DOI
  17. Ibarra-Molero B Loladze VV Makhatadze GI Sanchez-Ruiz JM 1999 Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability Biochemistry 38 8138 8149 10.1021/bi9905819 10387059 · DOI
  18. Jensen TJ Loo MA Pind S Williams DB Goldberg AL Riordan JR 1995 Multiple proteolytic systems, including the proteasome, contribute to CFTR processing Cell 83 129 135 10.1016/0092-8674(95)90241-4 7553864 · DOI
  19. Jiang L Mishra P Hietpas RT Zeldovich KB Bolon DN 2013 Latent effects of Hsp90 mutants revealed at reduced expression levels PLoS Genetics 9 e1003600 10.1371/journal.pgen.1003600 23825969 PMC3694843 · DOI
  20. Kellogg EH Leaver-Fay A Baker D 2011 Role of conformational sampling in computing mutation-induced changes in protein structure and stability Proteins 79 830 838 10.1002/prot.22921 21287615 PMC3760476 · DOI
  21. Kimura Y Yashiroda H Kudo T Koitabashi S Murata S Kakizuka A Tanaka K 2009 An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis Cell 137 549 559 10.1016/j.cell.2009.02.028 19410548 · DOI
  22. Koç A Wheeler LJ Mathews CK Merrill GF 2004 Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools The Journal of Biological Chemistry 279 223 230 10.1074/jbc.M303952200 14573610 · DOI
  23. Komander D Rape M 2012 The ubiquitin code Annual Review of Biochemistry 81 203 229 10.1146/annurev-biochem-060310-170328 22524316 · DOI · Paper page
  24. Kortemme T Baker D 2002 A simple physical model for binding energy hot spots in protein-protein complexes Proceedings of the National Academy of Sciences of the United States of America 99 14116 14121 10.1073/pnas.202485799 12381794 PMC137846 · DOI
  25. Lange OF Lakomek NA Farès C Schröder GF Walter KF Becker S Meiler J Grubmüller H Griesinger C de Groot BL 2008 Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution Science 320 1471 1475 10.1126/science.1157092 18556554 · DOI
  26. Lee DH Goldberg AL 1996 Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae The Journal of Biological Chemistry 271 27280 27284 10.1074/jbc.271.44.27280 8910302 · DOI
  27. Lee C Schwartz MP Prakash S Iwakura M Matouschek A 2001 ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular Cell 7 627 637 10.1016/S1097-2765(01)00209-X 11463387 · DOI
  28. Lee SY Pullen L Virgil DJ Castañeda CA Abeykoon D Bolon DN Fushman D Castaneda C.A 2014 Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function Journal of Molecular Biology 426 1377 1389 10.1016/j.jmb.2013.10.042 24361330 PMC3951674 · DOI
  29. Lindquist SL Kelly JW 2011 Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis Cold Spring Harbor Perspectives in Biology 3 10.1101/cshperspect.a004507 PMC3225948 21900404 · DOI
  30. Peng J Schwartz D Elias JE Thoreen CC Cheng D Marsischky G Roelofs J Finley D Gygi SP 2003 A proteomics approach to understanding protein ubiquitination Nature Biotechnology 21 921 926 10.1038/nbt849 12872131 · DOI
  31. Petermann E Orta ML Issaeva N Schultz N Helleday T 2010 Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair Molecular Cell 37 492 502 10.1016/j.molcel.2010.01.021 20188668 PMC2958316 · DOI
  32. Phillips AH Zhang Y Cunningham CN Zhou L Forrest WF Liu PS Steffek M Lee J Tam C Helgason E Murray JM Kirkpatrick DS Fairbrother WJ Corn JE 2013 Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling Proceedings of the National Academy of Sciences of the United States of Americ 110 11379 11384 10.1073/pnas.1302407110 PMC3710810 23801757 · DOI
  33. Powers ET Morimoto RI Dillin A Kelly JW Balch WE 2009 Biological and chemical approaches to diseases of proteostasis deficiency Annual Review of Biochemistry 78 959 991 10.1146/annurev.biochem.052308.114844 19298183 · DOI · Paper page
  34. Prakash S Tian L Ratliff KS Lehotzky RE Matouschek A 2004 An unstructured initiation site is required for efficient proteasome-mediated degradation Nature Structural & Molecular Biology 11 830 837 10.1038/nsmb814 15311270 · DOI · Paper page
  35. Reinke A Chen JC Aronova S Powers T 2006 Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p The Journal of Biological Chemistry 281 31616 31626 10.1074/jbc.M603107200 16923813 · DOI
  36. Rock KL Gramm C Rothstein L Clark K Stein R Dick L Hwang D Goldberg AL 1994 Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules Cell 78 761 771 10.1016/S0092-8674(94)90462-6 8087844 · DOI · Paper page
  37. Rockah-Shmuel L Tóth-Petróczy Á Tawfik DS Toth-Petroczy A 2015 Systematic mapping of protein mutational space by prolonged drift reveals the deleterious effects of seemingly neutral mutations PLoS Computational Biology 11 e1004421 10.1371/journal.pcbi.1004421 26274323 PMC4537296 · DOI
  38. Roscoe BP Thayer KM Zeldovich KB Fushman D Bolon DN 2013 Analyses of the effects of all ubiquitin point mutants on yeast growth rate Journal of Molecular Biology 425 1363 1377 10.1016/j.jmb.2013.01.032 23376099 PMC3615125 · DOI
  39. Roscoe BP Bolon DN 2014 Systematic exploration of ubiquitin sequence, E1 activation efficiency, and experimental fitness in yeast Journal of Molecular Biology 426 2854 2870 10.1016/j.jmb.2014.05.019 24862281 PMC4102620 · DOI
  40. Shabek N Iwai K Ciechanover A 2007 Ubiquitin is degraded by the ubiquitin system as a monomer and as part of its conjugated target Biochemical and Biophysical Research Communications 363 425 431 10.1016/j.bbrc.2007.08.185 17870054 · DOI
  41. Sharp PM Li WH 1987 Ubiquitin genes as a paradigm of concerted evolution of tandem repeats Journal of Molecular Evolution 25 58 64 10.1007/BF02100041 3041010 · DOI
  42. Sloper-Mould KE Jemc JC Pickart CM Hicke L 2001 Distinct functional surface regions on ubiquitin The Journal of Biological Chemistry 276 30483 30489 10.1074/jbc.M103248200 11399765 · DOI
  43. Stockwell BR 2000 Chemical genetics: ligand-based discovery of gene function Nature Reviews Genetics 1 116 125 10.1038/35038557 PMC3171795 11253651 · DOI
  44. Thrower JS Hoffman L Rechsteiner M Pickart CM 2000 Recognition of the polyubiquitin proteolytic signal The EMBO Journal 19 94 102 10.1093/emboj/19.1.94 10619848 PMC1171781 · DOI
  45. Vale RD DeRisi J Phillips R Mullins RD Waterman C Mitchison TJ 2012 Graduate education. Interdisciplinary graduate training in teaching labs Science 338 1542 1543 10.1126/science.1216570 23258877 PMC3810400 · DOI
  46. Wanke V Cameroni E Uotila A Piccolis M Urban J Loewith R De Virgilio C 2008 Caffeine extends yeast lifespan by targeting TORC1 Molecular Microbiology 69 277 285 10.1111/j.1365-2958.2008.06292.x 18513215 · DOI
  47. Wintrode PL Makhatadze GI Privalov PL 1994 Thermodynamics of ubiquitin unfolding Proteins 18 246 253 10.1002/prot.340180305 8202465 · DOI
  48. Xu P Duong DM Seyfried NT Cheng D Xie Y Robert J Rush J Hochstrasser M Finley D Peng J Xu P Xie Y 2009 Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation Cell 137 133 145 10.1016/j.cell.2009.01.041 19345192 PMC2668214 · DOI
  49. Zhang W Qin Z Zhang X Xiao W 2011 Roles of sequential ubiquitination of PCNA in DNA-damage tolerance FEBS Letters 585 2786 2794 10.1016/j.febslet.2011.04.044 21536034 · DOI
  50. Zolk O Schenke C Sarikas A 2006 The ubiquitin-proteasome system: focus on the heart Cardiovascular Research 70 410 421 10.1016/j.cardiores.2005.12.021 16497285 · DOI
  51. Zuin A Isasa M Crosas B 2014 Ubiquitin signaling: extreme conservation as a source of diversity Cells 3 690 701 10.3390/cells3030690 25014160 PMC4197634 · DOI

Cited by

Sorted by citations

Authors
Mavor, D., Barlow, K., Thompson, S., Barad, B. A., Bonny, A. R., Cario, C. L., Gaskins, G., Liu, Z., Deming, L., Axen, S. D., Caceres, E., Chen, W., Cuesta, A., Gate, R. E., Green, E. M., Hulce, K. R., Ji, W., Kenner, L. R., Mensa, B., Morinishi, L. S., Moss, S. M., Mravic, M., Muir, R. K., Niekamp, S., Nnadi, C. I., Palovcak, E., Poss, E. M., Ross, T. D., Salcedo, E. C., See, S. K., Subramaniam, M., Wong, A. W., Li, J., Thorn, K. S., Conchúir, S. Ó., Roscoe, B. P., Chow, E. D., DeRisi, J. L., Kortemme, T., Bolon, D. N., & Fraser, J. S.
Affiliations
  • University of California, San Francisco (US)
  • Howard Hughes Medical Institute (US)
  • Bioinformatics Graduate Group, University of California, San Francisco, San Francisco, United States
  • QB3 (US)
  • University of Massachusetts Chan Medical School (US)
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2016-04-25
Volume
5
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.15802
PMC
PMC4862753
OpenAlex
W2344038501
Cited by
89
References
3
Citations by year
2026: 12 · 2025: 5 · 2024: 9 · 2023: 8 · 2022: 7 · 2021: 7 · 2020: 10 · 2019: 10 · 2018: 9 · 2017: 9 · 2016: 3
FWCI
4.41
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion, cc-by-nc-nd)
  • eScholarship@UMassChan (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Science Foundation (Graduate Research Fellowships, DBI-1564692)
  • University of California, San Francisco (Ralph H Kellogg Endowed Chancellor's Fellowship)
  • National Institute of General Medical Sciences (Initiative for Maximizing Student Development Program)
  • National Institutes of Health (GM117189, GM112844)
  • NIH Office of the Director (OD009180)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
ubiquitin, fitness landscape, deep mutational scanning, chemical stress, small molecule perturbations, mutation tolerance, ubiquitin-proteasome system, hydrophobic patch, hygromycin B, DTT, buffering
MeSH terms
Biology, DNA Mutational Analysis, Humans, Saccharomyces cerevisiae, Stress, Physiological, Students, Universities, Ubiquitin, Proteasome Endopeptidase Complex, Mutant Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review