title: Establishment of a mutation system in Tetrahymena outer arm dynein and P-loop functions of the alpha heavy chain (Dyh3p).
abstract: Axonemal dyneins are large AAA+ type motor proteins that exhibit unique motor properties during ciliary beating. This study established a mutation system for Tetrahymena outer arm dynein and characterized four nucleotide-binding loops (P-loops; P1-P4) in the alpha heavy chain (Dyh3p). Macronuclear transformation of the mutant DYH3 genes in DYH3-knockout (KO-DYH3) cells enabled P-loop mutations that abolish the ability of nucleotide binding to be stably maintained in the polyploid genome. This mutation system revealed that the P3 and P4 mutant dyneins rescued lethality in macronuclear KO-DYH3 cells and exhibited normal ciliary localization. Intriguingly, however, an in vitro motility assay showed that the P3 mutation abolished the motor activity of Dyh3p, whereas the P4 mutation did not affect the gliding velocity or gliding index of Dyh3p. In contrast, no P1 or P2 mutant cells were isolated from the KO-DYH3 cells, which suggests that nucleotide binding at the P1 and P2 sites is required for the intracellular function of Dyh3p. This mutation system will be useful for further molecular studies of diverse axonemal dyneins and ciliary motility.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-0041, Japan. Electronic address: cedam@mail.ecc.u-tokyo.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Edamatsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D056444:Axonemal Dyneins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002923:Cilia / Q000378:metabolism; D055785:Gene Knockdown Techniques; D017125:Genes, Protozoan; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism*
chemical_list: D020409:Molecular Motor Proteins; D015800:Protozoan Proteins; D056444:Axonemal Dyneins
keywords: Ciliary motility; Ciliated protozoan; Cytoskeletal protein; Dynein; Tetrahymena