Lyons-Abbott Sally, Sackett Dan L, Wloga Dorota, Gaertig Jacek, Morgan Rachel E, Werbovetz Karl A, Morrissette Naomi S
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
Eukaryot Cell. 2010 Dec;9(12):1825-34. doi: 10.1128/EC.00140-10. Epub 2010 Sep 24.
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.
植物和原生动物的微管对二硝基苯胺具有选择性敏感性，而二硝基苯胺不会破坏脊椎动物或真菌的微管。嗜热四膜虫是二硝基苯胺敏感微管蛋白的丰富来源，我们已经对单一的嗜热四膜虫α-微管蛋白基因进行了改造，以创建仅在功能性二聚体中表达突变α-微管蛋白的菌株。先前的研究在人类寄生虫弓形虫中鉴定出多种赋予二硝基苯胺抗性的α-微管蛋白突变，当将其中两种突变（L136F和I252L）引入嗜热四膜虫时，它们在这些自由生活的纤毛虫中赋予了抗性。相对于野生型嗜热四膜虫微管蛋白，由L136F或I252Lα-微管蛋白组成的纯化微管蛋白异二聚体对二硝基苯胺oryzalin的亲和力降低。此外，相对于野生型嗜热四膜虫微管蛋白的特性，L136F替代显著降低了微管组装的临界浓度。我们的数据为α-微管蛋白上提议的二硝基苯胺结合位点提供了额外的支持，并验证了嗜热四膜虫用于表达遗传同质的突变微管蛋白群体以进行生化表征的用途。
