The cytoplasmic Cu,Zn superoxide dismutase of saccharomyces cerevisiae is required for resistance to freeze-thaw stress. Generation of free radicals during freezing and thawing.
The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor. Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected. This indicated that superoxide anions were formed during freezing and thawing. This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite. Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme. Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap. Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain.
Park
J I
JI
School of Biochemistry & Molecular Genetics and Cooperative Research Center for Food Industry Innovation, University of New South Wales, Sydney, New South Wales 2052, Australia.
Grant
C M
CM
Davies
M J
MJ
Dawes
I W
IW
D003300:Copper; D003593:Cytoplasm / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000235:genetics; D005615:Freezing; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005980:Glutathione Reductase / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology; D017382:Reactive Oxygen Species; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D013482:Superoxide Dismutase / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D014157:Transcription Factors / Q000235:genetics; D015032:Zinc
D004268:DNA-Binding Proteins; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C086677:YAP1 protein, S cerevisiae; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D005980:Glutathione Reductase; D005721:Glutamate-Cysteine Ligase; D015032:Zinc; D010100:Oxygen