Truncation of the carboxy-terminal domain of yeast beta-tubulin causes temperature-sensitive growth and hypersensitivity to antimitotic drugs

Abstract

beta-tubulin of budding yeast Saccharomyces cerevisiae is a polypeptide of 457 amino acids encoded by the unique gene TUB2. We investigated the function of the carboxy-terminal part of yeast beta-tubulin corresponding to the carboxy-terminal variable domain of mammalian and avian beta-tubulins. The GAA codon for Glu-431 of TUB2 was altered to TAA termination codon by using in vitro site-directed mutagenesis so that the 27-amino acid residues of the carboxyl terminus was truncated when expressed. The mutagenized TUB2 gene (tub2(T430)) was introduced into a haploid strain in which the original TUB2 gene had been disrupted. The tub2(T430) haploid strain grows normally less than 30 but not at 37 degrees C. The truncation of the carboxyl terminus caused hypersensitivity to antimitotic drugs and low spore viability at the permissive temperature for vegetative growth. Immunofluorescence labeling with antitubulin antibody and DNA staining with 4',6'-diamidino-2-phenylindole showed that in these cells at 37 degrees C, formation of spindle microtubules and nuclear division was inhibited and cytoplasmic microtubule distribution was aberrant. These results suggest that functions of the carboxy-terminal domain of yeast beta-tubulin are necessary for cells growing under suboptimal growth conditions although it is not essential for growth under the optimal growth conditions. Cells bearing tub2(411), a tub2 gene in which the GAA codon for Glu-412 was altered to TAA were no more viable at any temperature. In addition, a haploid strain carrying two functional beta-tubulin genes is not viable.