TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization.

Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation.

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional.

Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.

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.

Tubulin glycylation and glutamylation deficiencies in unconventional insect axonemes.

Though the 9+2 axonemal organization has generally been conserved throughout metazoan evolution, insect spermatozoa possess a substantial variety in axoneme ultrastructure, displaying different axonemal patterns. Therefore, insects provide a wide range of models that may be useful for the study of the mechanisms of axoneme assembly. We have used antibodies specific for glutamylated, monoglycylated, and polyglycylated tubulin to investigate the tubulin isoform content expressed in the unorthodox sperm axonemes of four insect species belonging to both of the superorders Palaeoptera and Neoptera.

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena.

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes.

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni.

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme.

Glutamylated tubulin: diversity of expression and distribution of isoforms.

Glutamylation of alpha and beta tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized.