, Volume 136, Issue 1, pp 28-38

Characterization of glyoxysomes in yeasts and their transformation into peroxisomes in response to changes in environmental conditions

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Abstract

During growth of the yeasts Candida utilis and Hansenula polymorpha in mineral media containing ethanol as a carbon source and ammonium sulphate as a nitrogen source, the specific activities of isocitrate lyase and malate synthase were significantly increased when compared to glucose/ammonium sulphate-grown cells. In addition to the enhanced levels of these glyoxylate cycle enzymes, an increase in the specific activities of d-amino acid oxidase, amine oxidase or urate oxidase was observed when ammonium sulphate in the ethanol medium was replaced by d-alanine, methyl- or ethylamine, or uric acid. The subcellular localization of these enzymes was investigated by cell fractionation studies involving homogenization of protoplasts followed by differential and sucrose gradient centrifugation. In ethanol/ammonium sulphate-grown cells, isocitrate lyase and malate synthase cosedimented in a fraction together with catalase and part of the malate dehydrogenase. Electron microscopy revealed that this fraction consisted of microbodies which must be regarded as glyoxysomes. Two other glyoxylate cycle enzymes, citrate synthase and aconitase together with the other part of malate dehydrogenase, cosedimented with cytochrome c oxidase, a mitochondrial marker enzyme. In ethanol/d-alanine-, ethanol/methylamine- or ethanol/ethylamine-grown C. utilis and ethanol/uric acid-grown H. polymorpha, a peroxisomal enzyme, i.e. d-amino acid oxidase, amine oxidase or uric acid oxidase cosedimented with the glyoxysomal key enzymes. Cytochemical staining experiments demonstrated that in these variously-grown cells the activities of the oxidases were confined to the microbodymatrix; this also contained malate synthase activity.

Transfer of C. utilis cells from glucose/ammonium sulphate- into ethanol/ammonium sulphate-containing media resulted in an increase in the original size and volume fraction of the microbodies. A further increase was observed when ammonium sulphate was replaced by methylamine. Essentially similar results were obtained with H. polymorpha cells. In neither of the two organisms indications of de novo synthesis of microbodies was obtained during transfer experiments. Invariably the microbodies developing in cells placed in the new environment originated from organelles already present in the inoculum cells by import of the substratespecific enzyme protein(s). The combined results of biochemical, cytochemical and electron microscopical experiments showed that in the yeasts studied under appropriate conditions glyoxysomal and peroxisomal enzyme activities were localized in one and the same microbody, rather than in separate organelles.