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.
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References
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
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
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
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
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
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
Novikoff AB, Beaufay H, de Duve C (1956) Electron microscopy of lysosome-rich fractions from rat liver. J Biophys Biochem Cytol 2(4):179
Leighton W (1964) Behavior of solutions of a linear differential equation of second order. PNAS 52(3):830–832
Veenhuis M, Mateblowski M, Kunau WH, Harder W (1987) Proliferation of microbodies in Saccharomyces cerevisiae. Yeast 3(2):77–84
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
Jones EW (1977) Proteinase mutants of Saccharomyces cerevisiae. Genetics 85(1):23–33
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
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
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
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
Zhu A, Romero R, Petty HR (2011) An enzymatic colorimetric assay for glucose-6-phosphate. Anal Biochem 419(2):266–270
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
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
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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
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DOI: https://doi.org/10.1007/978-1-0716-3048-8_22
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