Johnson V, et al. (2011) Design, overexpression, and purification of polymerization-blocked yeast a?-tubulin mutants. Biochemistry 50(40):8636-44
Abstract: Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of a?-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 a?-tubulin represents an obstacle to deeper insight into a?-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain a?-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 a?-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 a?-tubulin mutants. These "blocked" forms of a?-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 a?-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 a?-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of a?-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation.
|Status: Published||Type: Journal Article||PubMed ID: 21888381|
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