Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae.
The allele scd 1 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 CDC11. 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 sodl-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 SOD1 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 also fail to completely self-associate. Both phenotypes could be due to the inability of these polypeptides to adopt the native SOD-1 conformation.
Chang
E C
EC
Department of Biochemistry, School of Medicine and Biomedical Sciences, State of University of New York, Buffalo 14214.
Crawford
B F
BF
Hong
Z
Z
Bilinski
T
T
Kosman
D J
DJ
D000483:Alleles; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D002874:Chromosome Mapping; D003300:Copper / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D012331:RNA, Fungal; D012333:RNA, Messenger; D003300:Copper; D002374:Catalase; D013482:Superoxide Dismutase