title: α-Tubulin mutations alter oryzalin affinity and microtubule assembly properties to confer dinitroaniline resistance.
abstract: Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Sally
authors.initials: S
authors.lastname: Lyons-Abbott
authors.forename: Dan L
authors.initials: DL
authors.lastname: Sackett
authors.forename: Dorota
authors.initials: D
authors.lastname: Wloga
authors.forename: Jacek
authors.initials: J
authors.lastname: Gaertig
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Morgan
authors.forename: Karl A
authors.initials: KA
authors.lastname: Werbovetz
authors.forename: Naomi S
authors.initials: NS
authors.lastname: Morrissette
mesh_terms: D001665:Binding Sites; D003049:Coccidiostats / Q000494:pharmacology*; D004136:Dinitrobenzenes / Q000494:pharmacology*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D013424:Sulfanilamides / Q000494:pharmacology*; D016808:Tetrahymena thermophila / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
chemical_list: D003049:Coccidiostats; D004136:Dinitrobenzenes; D015800:Protozoan Proteins; D013424:Sulfanilamides; D014404:Tubulin; C012465:oryzalin