Functional characterization of lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p)

Highlights: • A mutation system for analyzing lethal mutations in Dyh3p was established. • The P1 mutant dynein localizes in the oral apparatus and the proximal region of the cilia. • The P2 mutant dynein localizes only in the oral apparatus. • The P1 and P2 mutations decrease the swimming and feeding of Tetrahymena cells. • The P1 and P2 mutant dyneins show similar microtubule-binding properties. Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3-knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility.