Functional consequences in yeast of single-residue alterations in a consensus calmodulin.
A synthetic gene encoding a 'consensus' calmodulin (synCaM) was able to substitute for the Saccharomyces cerevisiae calmodulin gene (CMDI), even though synCaM is only 60% identical in primary amino acid sequence to yeast Cmd1. Twelve different synCaM mutants were also expressed in yeast. Seven of the 12 mutant synCaMs supported germination and growth of Cmd1-deficient spores. Five of the 12 mutant synCaMs were incapable of supporting germination of Cmd1-deficient spores and, of these, four were also incapable of supporting vegetative growth of Cmd1-deficient haploid cells. The five nonfunctional synCaM mutants were expressed at levels equivalent to, or higher than, the seven synCaM mutants that were able to substitute for Cmd1; thus, the inability to function was not simply due to inadequate expression or rapid degradation. All nonfunctional synCaM mutants shared a single charge reversal mutation in the central helix (E84K), which was found to be sufficient to confer the lethal phenotype. The ability of another mutant synCaM (S101F) to support growth of Cmd1-deficient cells was dependent on cell ploidy. Another mutant (K115Y) supported spore germination and vegetative growth, but not meiosis and sporulation. The terminal phenotype of cells lacking a functional calmodulin included a dramatic accumulation of polymerized microtubules.
Harris
E
E
Department of Molecular and Cell Biology, University of California at Berkeley 94720-3202.
Watterson
D M
DM
Thorner
J
J
D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D016384:Consensus Sequence; D004563:Electrochemistry; D015870:Gene Expression; D005800:Genes, Fungal; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011003:Ploidies; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D013172:Spores, Fungal / Q000235:genetics / Q000502:physiology
D002147:Calmodulin