Truncation of the Carboxy-Terminal Domain of Yeast 13-Tubulin Causes Temperature-sensitive Growth and Hypersensitivity to Antimitotic Drugs

13-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 [I-tubulin corresponding to the carboxy-terminal variable domain of mammalian and avian fi-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 <30 but not at 37°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',6Miamidino-2-phenylindole showed that in these cells at 37°C, formation of spindle microtubules and nuclear division was inhibited and cytoplasmic microtubule distribution was aberrant. These results suggest that functions of the carboxyterminal domain of yeast 13-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 13-tubulin genes is not viable. M ICROTUBULES are ubiquitous, cellular structures in all eucaryotic cells, and are involved in a variety of functions including mitosis and cell motility (Dustin, 1984). Microtubules consist of heterodimeric subunits of aand 13-tubulin (Dustin, 1984). Both aand 13-tubulin are encoded by multigene families in higher eucaryotes, which generate isotypic variants of the proteins (Cleveland, 1987). The carboxy-terminal 15-amino acid residues of I~-tubulin isotypes are characteristically diversified whereas the rest of the amino acid sequence is highly conserved. Interestingly, the isotypic variation is well-conserved among mammals and avians including mouse, human, and chicken (Wang et al., 1986; Sullivan et al., 1986; Cleveland, 1987). This has prompted the assumption that the isotypic variation has functional significance. However, no direct evidence for this assumption has been provided experimentally. Furthermore, the carboxy-terminal sequence of 13-tubulin has been implicated in the regulation of tubulin polymerization perhaps by interacting with microtubule associated proteins (Serrano et al., 1984a). In this study, we investigated biological functions associated with the carboxy-terminal polypeptide of 13-tubulin using yeast Saccharomyces cerevisiae. S. cerevisiae have two ~tand one 13-tubulin genes in a haploid genome (Neff et al., E Matsuzaki's present address is Laboratory of Developmental and Molecular Biology, The Rockefeller University, New York, NY. 1983; Schatz et al., 1986a). Yeast 13-tubulin has 12 (or 13) amino acids more in its carboxyl terminus than its mammalian counterparts (Neff et al., 1983). Nevertheless, on the basis of comparison with mammalian 13-tubulin isotypes, the yeast polypeptide appears to be divided into two structural domains, the carboxy-terminal variable domain and the constant domain (see Fig. 1). In addition, chimeric 13-tubulin being composed of mammalian constant domain and yeast carboxy-terminal domain has recently been shown to be normally polymerized as microtubules in cultured mammalian cells (Bond et al., 1986). These observations suggest that the carboxyl terminus of yeast 13-tubulin could have similar functions to its mammalian counterpart. Using techniques of site-directed mutagenesis, we introduced a nonsense codon to yeast 13-tubulin gene just before the variable domain, and examined whether or not phenotypes of yeast cells expressing the truncated fl-tubulin polypeptide are altered. Materials and Methods