Eukaryot Cell. 2010 Sep 24. [Epub ahead of print]
{alpha}-tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties to Confer Dinitroaniline Resistance
Lyons-Abbott S, Sackett DL, Wloga D, Gaertig J, Morgan RE, Werbovetz KA, Morrissette NS.
Department of Molecular Biology and Biochemistry, University of CA, Irvine 92617; Laboratory of Integrative and Medical Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 20892; Department of Cellular Biology, University of Georgia, 30602; Division of Medicinal Chemistry & Pharmacognosy, Ohio State University, 43210.
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 provides additional support for the proposed dinitroaniline binding site on α-tubulin and validates the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.
PMID: 20870876
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