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 ␣-tubulin gene to create strains that solely express mutant ␣-tubulin in functional dimers. Previous research identified multiple ␣-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 ␣-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 ␣-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.An extraordinary number of small molecules target the eukaryotic ␣-␤ tubulin dimer. Compounds that shift the normal equilibrium between free dimers and polymers to destabilize or stabilize microtubules are exploited for diverse applications, ranging from cancer chemotherapy to treatment of helminth infections (31, 32). Although many compounds interact with almost all tubulin isotypes, some small molecules are selectively active against phylogenetically restricted subsets of tubulins. For example, helminth and fungal tubulins are selectively sensitive to several benzimidazoles (benomyl, albendazole, and mebendazole) that require the presence of "susceptible" amino acids (E198 and F200) in ␤-tubulin (33,36,37). Dinitroanilines represent another group of selective small molecules. They are synthetic compounds that inhibit microtubules in plants and protozoa but are inactive against the microtubules of vertebrates and fungi (reviewed in references 50, 53, and 70). These molecules (e.g., oryzalin and trifluralin) have been used in commercial herbicide formulations for over 40 years (53). Dinitroaniline binding studies using plant, protozoan, and vertebrate tubulins established that only sensitive tubulins bind dinitroanilines (9,28,49,72). The ability of dinitroanilines to selectively disrupt the microtubules of protozoan parasites without affecting vertebrate microtubules suggests the exciting possibility that we may be able to develop novel antiparasitic agents by understanding the mechanism of action of these compounds on sensitive tubulins.Resistance to microtubule-disrupting or -stabilizing drugs is often associated with point mutations to ␣-or ␤-tubulin that alter polymerization or binding site properties of tubulin heterodimers. Genetic studies of a wide variety of dinitroanilinesensitive organisms have identified ...

Salt Stress–Induced Disassembly ofArabidopsisCortical Microtubule Arrays Involves 26S Proteasome–Dependent Degradation of SPIRAL1

“…To assay the stability of cortical MTs in 26S proteasome mutants, we first tested the responses of rpn1a-4, rpt2a-2, rpn10-1, and rpn12a-1 mutants to the MT-destabilizing drugs oryzalin, which binds to a-tubulin, and propyzamide, which binds to b-tubulin (Nakamura et al, 2004;Lyons-Abbott et al, 2010). We showed previously that the total 26S proteasome activity in the four tested proteasome mutants is affected to different levels, with rpt2a-2 carrying the weakest and rpn10-1 the strongest defect in 26S proteasome function .…”

Pyridachlometyl has a novel anti‐tubulin mode of action which could be useful in anti‐resistance management

“…Among these herbicides, the binding pocket of carbamates, such as chlorpropham, and benzamides, such as pronamide, are assumed to be the colchicine binding site in β‐tubulin . However, dinitroanilines, such as oryzalin, trifluralin, and pendimethalin, have been reported to bind to α‐tubulin . Their binding site has been investigated mainly using protozoan organisms .…”

Dissecting the cellular functions of plant microtubules using mutant tubulins

“…Resistance to a MT‐depolymerizing drug may be caused by tubulin mutations that increase MT stability or decrease the MT's affinity for the drug. Although the TUA L136F mutant of T. gondii showed increased MT assembly in vitro [Lyons‐Abbott et al, ], several TUA mutations mapped to a distinct region of TUA, which may represent a binding site of dinitroanilines. Docking studies predict that dinitroanilines bind to a site directly behind the N‐loop, between helix 1 and β‐sheet 2 of TUA, and primarily destabilize the lateral contacts between PFs [Mitra and Sept, ].…”