Abstract
Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.
Highlights
Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue
Glutathione System in Peroxisomes—Our results demonstrated that His6-CbPmp20 had glutathione peroxidase (GPX) activity in vitro and that CbPmp20 exerted its physiological activity within peroxisomes
The most important fact revealed in this study is that CbPmp20 performs its physiological anti-oxidant function within peroxisomes as a GPX, which can use alkyl hydroperoxides and H2O2 as substrates
Summary
Candida boidinii Pmp (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. We detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii These results may indicate a physiological role for CbPmp as an antioxidant enzyme within peroxisomes rich in reactive oxygen species. The peroxisome is a ubiquitous organelle found in most eukaryotic cells in which various kinds of oxidative metabolism occur through at least one H2O2-generating peroxisomal oxidase [1, 2] These oxidative metabolisms yield reactive oxygen species (ROS), which can cause damage to all cellular constituents, i.e. nucleic acids, proteins, lipids, etc. Depletion of Pmp in S. cerevisiae did not affect its growth on oleate medium where normal peroxisomal metabolism is necessary for its growth. the binding of HsPmp to the PTS1 receptor Pex5p was shown, up to 50% of epitope-tagged HsPmp was detected in the cytosolic
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