Mutational analysis of cysteine residues in Saccharomyces cerevisiae beta-tubulin: Importance of Cys354 in microtubule dynamics

Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in beta-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae beta-tubulin individually to alanine and serine residues.;Of the twelve mutations, only three produced significant effects on cell phenotype, C12S, C354A and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation-produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were mis-shapen. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures) appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4°C the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. When the C12S and the two C354 mutations were made in a diploid strain the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains.;Mutation of C354 in beta-tubulin to either a serine or alanine residue produced phenotypes characteristic of microtubules with increased stability. To determine whether this was indeed the case, we used a GFP-Tub1p (alpha-tubulin) fusion construct to measure microtubule dynamics in vivo and purified yeast tubulin to measure microtubule dynamics in vitro . The mutations severely dampen dynamics in vivo and in vitro. In unbudded cells catastrophe and rescue frequencies were decreased by a factor of 40, dynamicity was only 1 to 2% of that in wild-type cells, and microtubules spent greater than 90% of the time attenuated. (Abstract shortened by UMI.).