A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele
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