Substitution at position 116 of Schizosaccharomyces pombe calmodulin decreases its stability under nitrogen starvation and results in a sporulation-deficient phenotype.
We constructed Schizosaccharomyces pombe strains that carry phenylalanine, instead of arginine, as residue 116 of calmodulin by site-directed mutagenesis of the cam1 gene. Whereas haploid strains carrying the mutant allele, designated cam1-F116, exhibit no defects in growth and mating, diploid strains homozygous for cam1-F116 are deficient in sporulation. The four nuclei generated by the two serial meiotic divisions are not encapsulated in these diploids. The mutation is recessive. Semiquantitative analysis using polyclonal antibodies showed that vegetatively growing cam1-F116 cells have a smaller amount of calmodulin than wild-type cells. The quantitative difference becomes more remarkable if the cells are starved for nitrogen, which is a condition for induction of sporulation. In addition to this in vivo observation, we showed in vitro that the mutant protein is susceptible to a proteolytic activity induced by nitrogen starvation that hardly affects the wild-type calmodulin. Thus, the sporulation deficiency of the cam1-F116 mutant may be ascribed to shortage of calmodulin due to proteolysis of the mutant molecules under nitrogen starvation. Two other mutations at position 116 resulted in similar but leakier Spo- phenotypes.
Takeda
T
T
Institute of Medical Science, University of Tokyo, Japan.
Imai
Y
Y
Yamamoto
M
M
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D010641:Phenotype; D004718:Saccharomycetales / Q000502:physiology*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013172:Spores, Fungal / Q000502:physiology; D014170:Transformation, Genetic
D002147:Calmodulin; D009584:Nitrogen