title: Design, overexpression, and purification of polymerization-blocked yeast αβ-tubulin mutants.
abstract: 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.
authors: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
authors: Vinu
authors: V
authors: Johnson
authors: Pelin
authors: P
authors: Ayaz
authors: Patrick
authors: P
authors: Huddleston
authors: Luke M
authors: LM
authors: Rice
mesh_terms: D019281:Dimerization; D015870:Gene Expression; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D058105:Polymerization; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C475498:Tub1 protein, S cerevisiae; C568590:Tub2 protein, S cerevisiae