Skip to main content
SpringerLink
Log in

Yeast-Based Screens to Target Alpha-Synuclein Toxicity

  • Protocol
  • First Online:
Alpha-Synuclein

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1948))

Abstract

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has been a remarkable experimental model for the discovery of fundamental biological processes. The high degree of conservation of cellular and molecular processes between the budding yeast and higher eukaryotes has made it a valuable system for the investigation of the molecular mechanisms behind various types of devastating human pathologies. Genetic screens in yeast provided important insight into the toxic mechanisms associated with the accumulation of misfolded proteins. Thus, using yeast genetics and high-throughput screens, novel molecular targets with therapeutic potential have been identified. Here, we describe a yeast screen protocol for the identification of genetic modifiers of alpha-synuclein (aSyn) toxicity, thereby accelerating the identification of novel potential targets for intervention in Parkinson’s disease (PD) and other synucleinopathies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Giorgini F, Muchowski PJ (2006) Screening for genetic modifiers of amyloid toxicity in yeast. Methods Enzymol 412:201–222

    Article  CAS  Google Scholar 

  2. Tenreiro S, Outeiro TF (2010) Simple is good: yeast models of neurodegeneration. FEMS Yeast Res 10:970–979

    Article  CAS  Google Scholar 

  3. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H et al (1996) Life with 6000 genes. Science 274:563–567

    Article  Google Scholar 

  4. Coller J, Parker R (2004) Eukaryotic mRNA decapping. Annu Rev Biochem 73:861–890

    Article  CAS  Google Scholar 

  5. Tsukuda T, Fleming AB, Nickoloff JA, Osley MA (2005) Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature 438:379–383

    Article  CAS  Google Scholar 

  6. Botstein D, Fink GR (2011) Yeast: an experimental organism for 21st century biology. Genetics 189:695–704

    Article  CAS  Google Scholar 

  7. Mager WH, Winderickx J (2005) Yeast as a model for medical and medicinal research. Trends Pharmacol Sci 26:265–273

    Article  CAS  Google Scholar 

  8. Carmona-Gutierrez D, Eisenberg T, Buttner S, Meisinger C, Kroemer G, Madeo F (2010) Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 17:763–773

    Article  CAS  Google Scholar 

  9. Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116:153–166

    Article  CAS  Google Scholar 

  10. Rinaldi T, Dallabona C, Ferrero I, Frontali L, Bolotin-Fukuhara M (2010) Mitochondrial diseases and the role of the yeast models. FEMS Yeast Res 10:1006–1022

    Article  CAS  Google Scholar 

  11. Goeckeler JL, Brodsky JL (2010) Molecular chaperones and substrate ubiquitination control the efficiency of endoplasmic reticulum-associated degradation. Diabetes Obes Metab 12:32–38

    Article  CAS  Google Scholar 

  12. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175

    Article  CAS  Google Scholar 

  13. Brodsky JL, Skach WR (2011) Protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems. Curr Opin Cell Biol 23:464–475

    Article  CAS  Google Scholar 

  14. Outeiro TF, Lindquist S (2003) Yeast cells provide insight into α-synuclein biology and pathobiology. Science 302:1772–1775

    Article  CAS  Google Scholar 

  15. Willingham S, Outeiro TF, DeVit MJ, Lindquist SL, Muchowski PJ (2003) Yeast genes that enhance the toxicity of a mutant huntingtin fragment or α-synuclein. Science 302:1769–1772

    Article  CAS  Google Scholar 

  16. Dixon C, Mathias N, Zweig RM, Davis DA, Gross DS (2005) α-Synuclein targets the plasma membrane via the secretory pathway and induces toxicity in yeast. Genetics 170:47–59

    Article  CAS  Google Scholar 

  17. Flower TR, Chesnokova LS, Froelich CA, Dixon C, Witt SN (2005) Heat shock prevents α-synuclein-induced apoptosis in a yeast model of Parkinson’s disease. J Mol Biol 351:1081–1100

    Article  CAS  Google Scholar 

  18. Buttner S, Bitto A, Ring J, Augsten M, Zabrocki P, Eisenberg T et al (2008) Functional mitochondria are required for α-synuclein toxicity in aging yeast. J Biol Chem 283:7554–7560

    Article  Google Scholar 

  19. Zabrocki P, Bastiaens I, Delay C, Bammens T, Ghillebert R, Pellens K et al (2008) Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson. Biochim Biophys Acta 1783:1767–1780

    Article  CAS  Google Scholar 

  20. Puccio H, Koenig M (2000) Recent advances in the molecular pathogenesis of Friedreich ataxia. Hum Mol Genet 9:887–892

    Article  CAS  Google Scholar 

  21. Outeiro TF, Muchowski PJ (2004) Molecular genetics approaches in yeast to study amyloid diseases. J Mol Neurosci 23:49–60

    Article  CAS  Google Scholar 

  22. Khurana V, Lindquist S (2010) Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker’s yeast? Nat Rev Neurosci 11:436–449

    Article  CAS  Google Scholar 

  23. Entian KD, Kötter P (2007) Yeast genetic strain and plasmid collections. In: Stansfield I, MJR S (eds) Yeast gene analysis, Methods in microbiology, vol 36, 2nd edn. Academic Press, New York, pp 629–666

    Google Scholar 

  24. Miller-Fleming L, Giorgini F, Outeiro TF (2008) Yeast as a model for studying human neurodegenerative disorders. Biotechnol J 3:325–338

    Article  CAS  Google Scholar 

  25. Karathia H, Vilaprinyo E, Sorribas A, Alves R (2011) Saccharomyces cerevisiae as a model organism: a comparative study. PLoS One 6:e16015

    Article  CAS  Google Scholar 

  26. Tenreiro S, Munder MC, Alberti S, Outeiro TF (2013) Harnessing the power of yeast to unravel the molecular basis of neurodegeneration. J Neurochem 127:438–452

    Article  CAS  Google Scholar 

  27. Menezes R, Tenreiro S, Macedo D, Santos CN, Outeiro TF (2015) From the baker to the bedside: yeast models of Parkinson’s disease. Microb Cell 2:262–279

    Article  CAS  Google Scholar 

  28. Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, Hill KJ et al (2009) Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity. Nat Genet 41:308–315

    Article  CAS  Google Scholar 

  29. Miller-Fleming L, Antas P, Pais TF, Smalley JL, Giorgini F, Outeiro TF (2014) Yeast DJ-1 superfamily members are required for diauxic-shift reprogramming and cell survival in stationary phase. Proc Natl Acad Sci U S A 111:7012–7017

    Article  CAS  Google Scholar 

  30. Zondler L, Miller-Fleming L, Repici M, Goncalves S, Tenreiro S, Rosado-Ramos R et al (2014) DJ-1 interactions with alpha-synuclein attenuate aggregation and cellular toxicity in models of Parkinson’s disease. Cell Death Dis 5:e1350

    Article  CAS  Google Scholar 

  31. Dhungel N, Eleuteri S, Li LB, Kramer NJ, Chartron JW, Spencer B et al (2015) Parkinson’s disease genes VPS35 and EIF4G1 interact genetically and converge on alpha-synuclein. Neuron 85:76–87

    Article  CAS  Google Scholar 

  32. Xiong Y, Coombes CE, Kilaru A, Li X, Gitler AD, Bowers WJ et al (2010) GTPase activity plays a key role in the pathobiology of LRRK2. PLoS Genet 6:e1000902

    Article  Google Scholar 

  33. Pereira C, Miguel ML, Saraiva L (2014) LRRK2, but not pathogenic mutants, protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model. Biochim Biophys Acta 1840:2025–2031

    Article  CAS  Google Scholar 

  34. Pereira C, Costa V, Martins LM, Saraiva L (2015) A yeast model of the Parkinson’s disease-associated protein Parkin. Exp Cell Res 333:73–79

    Article  CAS  Google Scholar 

  35. Buttner S, Delay C, Franssens V, Bammens T, Ruli D, Zaunschirm S et al (2010) Synphilin-1 enhances alpha-synuclein aggregation in yeast and contributes to cellular stress and cell death in a Sir2-dependent manner. PLoS One 5:e13700

    Article  Google Scholar 

  36. Tenreiro S, Rosado-Ramos R, Gerhardt E, Favretto F, Magalhaes F, Popova B et al (2016) Yeast reveals similar molecular mechanisms underlying alpha- and beta-synuclein toxicity. Hum Mol Genet 25:275–290

    Article  CAS  Google Scholar 

  37. Su LJ, Auluck PK, Outeiro TF, Yeger-Lotem E, Kritzer JA, Tardiff DF et al (2010) Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson’s disease models. Dis Model Mech 3:194–208

    Article  CAS  Google Scholar 

  38. Petroi D, Popova B, Taheri-Talesh N, Irniger S, Shahpasandzadeh H, Zweckstetter M, Outeiro TF, Braus GH (2012) Aggregate clearance of α-synuclein in Saccharomyces cerevisiae depends more on autophagosome and vacuole function than on the proteasome. J Biol Chem 287:27567–27579

    Article  CAS  Google Scholar 

  39. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J et al (2003) Alpha-Synuclein locus triplication causes Parkinson’s disease. Science 302:841

    Article  CAS  Google Scholar 

  40. Ibanez P, Bonnet AM, Debarges B, Lohmann E, Tison F, Pollak P et al (2004) Causal relation between alpha-synuclein gene duplication and familial Parkinson’s disease. Lancet 364:1169–1171

    Article  CAS  Google Scholar 

  41. Franssens V, Boelen E, Anandhakumar J, Vanhelmont T, Buttner S, Winderickx J (2010) Yeast unfolds the road map toward α-synuclein-induced cell death. Cell Death Differ 17:746–753

    Article  CAS  Google Scholar 

  42. Franssens V, Bynens T, Van den Brande J, Vandermeeren K, Verduyckt M, Winderickx J (2013) The benefits of humanized yeast models to study Parkinson’s disease. Oxidative Med Cell Longev 2013:760629

    Article  CAS  Google Scholar 

  43. Flower TR, Clark-Dixon C, Metoyer C, Yang H, Shi R, Zhang Z et al (2007) YGR198w (YPP1) targets A30P α-synuclein to the vacuole for degradation. J Cell Biol 177:1091–1104

    Article  CAS  Google Scholar 

  44. Gitler AD, Bevis BJ, Shorter J, Strathearn KE, Hamamichi S, Su LJ et al (2008) The Parkinson’s disease protein α-synuclein disrupts cellular Rab homeostasis. Proc Natl Acad Sci U S A 105:145–150

    Article  CAS  Google Scholar 

  45. Soper JH, Roy S, Stieber A, Lee E, Wilson RB, Trojanowski JQ et al (2008) α-Synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae. Mol Biol Cell 19:1093–1103

    Article  CAS  Google Scholar 

  46. Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B et al (2006) Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313:324–328

    Article  CAS  Google Scholar 

  47. Yeger-Lotem E, Riva L, Su LJ, Gitler AD, Cashikar AG, King OD et al (2009) Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nat Genet 41:316–323

    Article  CAS  Google Scholar 

  48. Zabrocki P, Pellens K, Vanhelmont T, Vandebroek T, Griffioen G, Wera S et al (2005) Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast. FEBS J 272:1386–1400

    Article  CAS  Google Scholar 

  49. Sampaio-Marques B, Felgueiras C, Silva A, Rodrigues M, Tenreiro S, Franssens V et al (2012) SNCA (alpha-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated mitophagy. Autophagy 8:1494–1509

    Article  CAS  Google Scholar 

  50. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906

    Article  CAS  Google Scholar 

  51. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S et al (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391

    Article  CAS  Google Scholar 

  52. Mnaimneh S, Davierwala AP, Haynes J, Moffat J, Peng WT, Zhang W, Yang X et al (2004) Exploration of essential gene functions via titratable promoter alleles. Cell 118:31–44

    Article  CAS  Google Scholar 

  53. Schuldiner M, Collins SR, Thompson NJ, Denic V, Bhamidipati A, Punna T, Ihmels J et al (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123:507–519

    Article  CAS  Google Scholar 

  54. Li Z, Vizeacoumar FJ, Bahr S, Li J, Warringer J, Vizeacoumar FS, Min R et al (2011) Systematic exploration of essential yeast gene function with temperature-sensitive mutants. Nat Biotechnol 29:361–367

    Article  CAS  Google Scholar 

  55. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK (2003) Global analysis of protein localization in budding yeast. Nature 425:686–691

    Article  CAS  Google Scholar 

  56. Gavin AC, Bösche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J et al (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147

    Article  CAS  Google Scholar 

  57. Gietz RD, Schiestl RH (1996) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269

    Google Scholar 

Download references

Acknowledgments

T.F.O. and G.H.B. are supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 721802.

Author information

Authors and Affiliations

  1. Department of Experimental Neurodegeneration, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Goettingen, Goettingen, Germany

    Inês Caldeira Brás & Tiago F. Outeiro

  2. Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, Göttingen, Germany

    Blagovesta Popova & Gerhard H. Braus

  3. Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Goettingen, Germany

    Blagovesta Popova, Gerhard H. Braus & Tiago F. Outeiro

  4. Max Planck Institute for Experimental Medicine, Goettingen, Germany

    Tiago F. Outeiro

Authors
  1. Inês Caldeira Brás
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Blagovesta Popova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gerhard H. Braus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tiago F. Outeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tiago F. Outeiro .

Editor information

Editors and Affiliations

  1. Dementia Research Institute, University College London, London, UK

    Tim Bartels

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Brás, I.C., Popova, B., Braus, G.H., Outeiro, T.F. (2019). Yeast-Based Screens to Target Alpha-Synuclein Toxicity. In: Bartels, T. (eds) Alpha-Synuclein. Methods in Molecular Biology, vol 1948. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9124-2_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9124-2_12

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9123-5

  • Online ISBN: 978-1-4939-9124-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation