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Detection of Ubiquitinated Peroxisomal Proteins in Yeast

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Peroxisomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1595))

Abstract

Ubiquitination is involved in different aspects of peroxisome formation, maintenance, and degradation. Consequently, simple methods for detecting ubiquitinated peroxisomal proteins are extremely useful in peroxisomal research. Here, we describe an immunoprecipitation-based technique that can be used to assess peroxisomal protein ubiquitination in yeast.

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References

  1. Fang S, Weissman AM (2004) A field guide to ubiquitylation. Cell Mol Life Sci 61(13):1546–1561

    Article  CAS  PubMed  Google Scholar 

  2. Williams C, van der Klei IJ (2013) Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin–proteasome system. Biochem Biophys Res Commun 438(2):395–401

    Article  CAS  PubMed  Google Scholar 

  3. Kragt A, Voorn-Brouwer T, van den Berg M, Distel B (2005) The Saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells. J Biol Chem 280(9):7867–7874

    Article  CAS  PubMed  Google Scholar 

  4. Leon S, Zhang L, McDonald WH, Yates J 3rd, Cregg JM, Subramani S (2006) Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation. J Cell Biol 172(1):67–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Platta HW, Girzalsky W, Erdmann R (2004) Ubiquitination of the peroxisomal import receptor Pex5p. Biochem J 384(1):37–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lingard MJ, Monroe-Augustus M, Bartel B (2009) Peroxisome-associated matrix protein degradation in Arabidopsis. Proc Natl Acad Sci U S A 106(11):4561–4566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Purdue PE, Lazarow PB (2001) Pex18p is constitutively degraded during peroxisome biogenesis. J Biol Chem 276(50):47684–47689

    Article  CAS  PubMed  Google Scholar 

  8. Hagstrom D, Ma C, Guha-Polley S, Subramani S (2014) The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20. Mol Biol Cell 25(17):2634–2643

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kim DY, Scalf M, Smith LM, Vierstra RD (2013) Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis. Plant Cell 25(5):1523–1540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B, Baldi P, Huang L, Kaiser P (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol Cell Proteomics 5(4):737–748

    Article  CAS  PubMed  Google Scholar 

  11. Seyfried NT, Xu P, Duong DM, Cheng D, Hanfelt J, Peng J (2008) Systematic approach for validating the ubiquitinated proteome. Anal Chem 80(11):4161–4169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim W, Bennett EJ, Huttlin EL, Guo A, Li J, Possemato A, Sowa ME, Rad R, Rush J, Comb MJ, Harper JW, Gygi SP (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44(2):325–340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Williams C, van den Berg M, Sprenger RR, Distel B (2007) A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J Biol Chem 282(31):22534–22543

    Article  CAS  PubMed  Google Scholar 

  14. Kragt A, Benne R, Distel B (2006) Ubiquitin: a new player in the peroxisome field. In: Mayer RJ, Ciechanover A, Rechsteiner M (eds) Protein degradation: cell biology of the ubiquitin-proteasome system, Protein degradation, vol 3. Wiley-VCH Verlag GmbH, Weinheim, Germany, pp 1–20

    Chapter  Google Scholar 

  15. El Magraoui F, Schrotter A, Brinkmeier R, Kunst L, Mastalski T, Muller T, Marcus K, Meyer HE, Girzalsky W, Erdmann R, Platta HW (2014) The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor. PLoS One 9(8):e105894

    Article  PubMed  PubMed Central  Google Scholar 

  16. El Magraoui F, Brinkmeier R, Schrotter A, Girzalsky W, Muller T, Marcus K, Meyer HE, Erdmann R, Platta HW (2013) Distinct ubiquitination cascades act on the peroxisomal targeting signal type 2 co-receptor Pex18p. Traffic 14(12):1290–1301

    Article  CAS  PubMed  Google Scholar 

  17. Liu X, Subramani S (2013) Unique requirements for mono- and polyubiquitination of the peroxisomal targeting signal co-receptor, Pex20. J Biol Chem 288(10):7230–7240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bellu AR, Salomons FA, Kiel JA, Veenhuis M, Van Der Klei IJ (2002) Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha. J Biol Chem 277(45):42875–42880

    Article  CAS  PubMed  Google Scholar 

  19. Pringle J, Adams A, Drubin D, Haarer B (1991) Immunofluorescence methods for yeast. Methods Enzymol 194:565–602

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by a VIDI grant (723.013.004) from the Netherlands Organization for Scientific Research (NWO), awarded to C.W.

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Authors and Affiliations

  1. Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9700 CC, Groningen, The Netherlands

    Natasha Danda & Chris Williams

Authors
  1. Natasha Danda
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  2. Chris Williams
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Corresponding author

Correspondence to Chris Williams .

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Editors and Affiliations

  1. College of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, United Kingdom

    Michael Schrader

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Danda, N., Williams, C. (2017). Detection of Ubiquitinated Peroxisomal Proteins in Yeast. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 1595. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6937-1_22

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  • DOI: https://doi.org/10.1007/978-1-4939-6937-1_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6935-7

  • Online ISBN: 978-1-4939-6937-1

  • eBook Packages: Springer Protocols

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