Design, overexpression, and purification of polymerization-blocked yeast αβ-tubulin mutants.

Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αβ-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αβ-tubulin represents an obstacle to deeper insight into αβ-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αβ-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αβ-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αβ-tubulin mutants. These "blocked" forms of αβ-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αβ-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αβ-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αβ-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation.

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