Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 1991-03, Vol.266 (7), p.4417-4424
• Main Authors: Chang, E.C. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Crawford, B.F, Hong, Z, Bilinski, T, Kosman, D.J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Alleles; amino acid sequence; Base Sequence; Blotting, Northern; Blotting, Western; Catalase - metabolism; Chromosome Mapping; Copper - metabolism; EXPRESION GENICA; EXPRESSION DES GENES; GENE; GENES; Genetic Complementation Test; loci; Molecular Sequence Data; MUTANT; MUTANTES; Mutation; nucleotide sequence; restriction endonuclease mapping; RNA, Fungal - genetics; RNA, Messenger - genetics; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; superoxide dismutase; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; SUPEROXIDO DISMUTASA; SUPEROXYDE DISMUTASE; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(20)64338-1

The allele scd1 is a recessive chromosomal mutation in Saccharomyces cerevisiae that eliminates Cu,Zn superoxide dismutase (SOD-1) activity. SOD-1- strains are unable to grow in 100% O2 in rich medium and are methionine and lysine auxotrophic when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). In this report, scd1 was genetically mapped to the right arm of chromosome X, 11 centimorgans proximal to cdc11. The gene for SOD-1 (SOD1) was physically mapped by Southern blot to restriction fragments containing CBC11. scd1 failed to complement a complete deletion of SOD1. Thus, scd1 maps to the SOD1 locus and is designated sod1-1. The molecular basis for the lack of SOD-1 activity in sod1-1 carrying strains has also been established. The size and amount of SOD-1 mRNA in the mutant were essentially the same as in wild type cells. Western blot analysis showed that the SOD-1 dimer and 16-kilodalton subunit that co-migrated electrophoretically with wild type yeast SOD-1 were abundant in Mutant cell extracts. However, two additional SOD-1 immunoreactive polypeptides were detected in these extracts in both denaturing and nondenaturing gels. None of the SOD-1 immunoreactive species in the mutant extracts exhibited superoxide dismutase activity. Transformants of the mutant strain carrying episomal, wild type SO-1D expressed wild type, active SOD-1 protein, indicating that the mutant allele had no discernible effect on the correct synthesis and activation of apoSOD-1. Size exclusion chromatography of soluble cell extracts derived from wild type and SOD1 deletion strains identified a copper binding peak that corresponded to SOD-1. This copper-binding fraction was absent in cell extracts from the sod1-1-containing strain although Western blot analysis of the corresponding chromatographic fractions showed that SOD-1 polypeptide was present in these fractions. Sequence data derived from the cloned genes showed that sod1-1 differed from SOD1 only in the adjacent 5'-noncoding region. The biochemical data indicate that this genetic alteration results in the synthesis of a collection of SOD-1 polypeptides that fail to bind copper and may 8% fail to completely self-associate. Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation