title: Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.
abstract: Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the alpha-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of alpha-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as alpha-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.
authors.affiliation: Department of Cellular Biology, 724 Biological Sciences Building, Athens GA 30602-2607, USA.
authors.forename: Dorota
authors.initials: D
authors.lastname: Wloga
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Rogowski
authors.forename: Neeraj
authors.initials: N
authors.lastname: Sharma
authors.forename: Juliette
authors.initials: J
authors.lastname: Van Dijk
authors.forename: Carsten
authors.initials: C
authors.lastname: Janke
authors.forename: Bernard
authors.initials: B
authors.lastname: Eddé
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Bré
authors.forename: Nicolette
authors.initials: N
authors.lastname: Levilliers
authors.forename: Virginie
authors.initials: V
authors.lastname: Redeker
authors.forename: Jianming
authors.initials: J
authors.lastname: Duan
authors.forename: Martin A
authors.initials: MA
authors.lastname: Gorovsky
authors.forename: Maria
authors.initials: M
authors.lastname: Jerka-Dziadosz
authors.forename: Jacek
authors.initials: J
authors.lastname: Gaertig
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002923:Cilia / Q000378:metabolism / Q000648:ultrastructure; D018698:Glutamic Acid / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D016808:Tetrahymena thermophila / Q000378:metabolism* / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism*
chemical_list: D014404:Tubulin; D018698:Glutamic Acid; D008025:Ligases; D010453:Peptide Synthases; C112698:tubulin polyglutamylase