Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene.
Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe-->Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl2 in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd 1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe-->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks.
Ohya
Y
Y
Department of Genetics, Stanford University School of Medicine, California 94305-5120.
Botstein
D
D
D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005800:Genes, Fungal; D005804:Genes, Lethal; D005808:Genes, Recessive; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship
D002147:Calmodulin; D005656:Fungal Proteins; D010649:Phenylalanine