Skip to main content

Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of β-Oxidation Activity

  • Protocol
  • First Online:
Peroxisomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2643))

  • 687 Accesses

Abstract

Subcellular fractionation approaches have allowed for the identification of various functionally distinct organelles including peroxisomes. The methods enable enrichment of organelles and combined with downstream assays allow for the identification of biochemical functions, composition, and structural characteristics of these compartments. In this chapter, we describe the methods for differential centrifugation and Nycodenz gradients in the yeast Saccharomyces cerevisiae and describe assays for fatty acid β-oxidation in intact cells and in peroxisomal fractions.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 229.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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. Dommes V, Baumgart C, Kunau WH (1981) Degradation of unsaturated fatty acids in peroxisomes. Existence of a 2,4-dienoyl-CoA reductase pathway. J Biol Chem 256(16):8259–8262

    Article  CAS  PubMed  Google Scholar 

  2. van Roermund CWT, Elgersma Y, Singh N, Wanders RJ, Tabak HF (1995) The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions. EMBO J 14(14):3480–3486

    Article  PubMed  PubMed Central  Google Scholar 

  3. Al-Saryi NA, Al-Hejjaj MY, van Roermund CWT, Hulmes GE, Ekal L, Payton C et al (2017) Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae. Sci Rep 7(1):1–9

    Article  CAS  Google Scholar 

  4. Hettema EH, van Roermund CWT, Distel B, van den Berg M, Vilela C, Rodrigues-Pousada C et al (1996) The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae. EMBO J 15(15):3813–3822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shani N, Watkins PA, Valle D (1995) PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene. Proc Natl Acad Sci U S A 92(13):6012–6016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Appelmans F, de Duve C (1955) Tissue fractionation studies. 3. Further observations on the binding of acid phosphatase by rat-liver particles. Biochem J 59(3):426–433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Novikoff AB, Beaufay H, de Duve C (1956) Electron microscopy of lysosome-rich fractions from rat liver. J Biophys Biochem Cytol 2(4):179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Leighton W (1964) Behavior of solutions of a linear differential equation of second order. PNAS 52(3):830–832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Veenhuis M, Mateblowski M, Kunau WH, Harder W (1987) Proliferation of microbodies in Saccharomyces cerevisiae. Yeast 3(2):77–84

    Article  CAS  PubMed  Google Scholar 

  10. Erdmann R, Blobel G (1995) Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J Cell Biol 128(4):509–523

    Article  CAS  PubMed  Google Scholar 

  11. Jones EW (1977) Proteinase mutants of Saccharomyces cerevisiae. Genetics 85(1):23–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. van Roermund CWT, Ijlst L, Baker A, Wanders RJA, Theodoulou FL, Waterham HR (2021) The Saccharomyces cerevisiae ABC subfamily D transporter Pxa1/Pxa2p co-imports CoASH into the peroxisome. FEBS Let 595(6):763–772

    Article  Google Scholar 

  13. Wanders RJA, Ijlst L, van Gennip AH, Jakobs C, de Jager JP, Dorland L et al (1990) Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid β-oxidation. J Inherit Metab Dis 13(3):311–314

    Article  CAS  PubMed  Google Scholar 

  14. van Roermund CWT, Hettema EH, van den Berg M, Tabak HF, Wanders RJA (1999) Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p. EMBO J 18(21):5843–5852

    Article  PubMed  PubMed Central  Google Scholar 

  15. Verleur N, Hettema EH, van Roermund CWT, Tabak HF, Wanders RJA (1997) Transport of activated fatty acids by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system. Eur J Biochem 249(3):657–661

    Article  CAS  PubMed  Google Scholar 

  16. Zhu A, Romero R, Petty HR (2011) An enzymatic colorimetric assay for glucose-6-phosphate. Anal Biochem 419(2):266–270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. van Roermund CWT, Drissen R, van den Berg M, Ijlst L, Hettema EH, Tabak HF et al (2001) Identification of a peroxisomal ATP carrier required for medium-chain fatty acid β-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae. Mol Cell Biol 21(13):4321–4329

    Article  PubMed  PubMed Central  Google Scholar 

  18. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P et al (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2):115–132

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Carlo van Roermund .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

van Roermund, C., Hettema, E. (2023). Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of β-Oxidation Activity. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 2643. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3048-8_22

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3048-8_22

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3047-1

  • Online ISBN: 978-1-0716-3048-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics