title: Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco.
abstract: Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Takashi
authors.identifier: http://orcid.org/0000-0002-9497-0950
authors.initials: T
authors.lastname: Hotta
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujita
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Seiichi
authors.initials: S
authors.lastname: Uchimura
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Masahiro
authors.initials: M
authors.lastname: Noguchi
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Taku
authors.initials: T
authors.lastname: Demura
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.).
authors.forename: Etsuko
authors.identifier: http://orcid.org/0000-0001-5812-1436
authors.initials: E
authors.lastname: Muto
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan (T.Ho., S.F., M.N., T.D., T.Ha.); andLaboratory of Molecular Biophysics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan (S.U., E.M.) hasimoto@bs.naist.jp.
authors.forename: Takashi
authors.identifier: http://orcid.org/0000-0002-8398-5479
authors.initials: T
authors.lastname: Hashimoto
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D011523:Protoplasts / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013535:Suspensions; D013552:Swine; D013912:Threonine / Q000737:chemistry; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism*; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; C109414:STU2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013535:Suspensions; D014404:Tubulin; D013912:Threonine