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. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

A posttranslational modification was detected in the carboxyl-terminal region of axonemal tubulin from Paramecium. Tubulin carboxyl-terminal peptides were isolated and analyzed by Edman degradation sequencing, mass spectrometry, and amino acid analysis. All of the peptides, derived from both alpha and beta tubulin subunits, were modified by polyglycylation, containing up to 34 glycyl units covalently bound to the gamma carboxyl group of glutamyl residues. This modification, present in one of the most stable microtubular systems, may influence microtubule stability or axoneme function, or both.

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. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of α- and β-tubulin, respectively. Mutants lacking all polyglycylation sites on α-tubulin have normal phenotype, whereas similar sites on β-tubulin are essential. A viable mutant with three mutated sites in β-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on β-tubulin were mutated were viable if they were cotransformed with an α-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of β-tubulin. In this double mutant, β-tubulin lacked detectable polyglycylation, while the α-β tubulin chimera was hyperglycylated compared with α-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of β-tubulin can be transferred to α-tubulin, indicating it is the total amount of polyglycylation on both α- and β-tubulin that is essential for survival.

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 ␣-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of ␣-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 ␣-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 ␣-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.The principal components of microtubules, heterodimers of ␣-and ␤-tubulin, are known to undergo several types of conserved posttranslational modifications (PTMs), including acetylation, detyrosination, phosphorylation, palmitoylation, glutamylation, and glycylation (61). Some of these PTMs strongly influence interactions between microtubules and microtubuleassociated proteins, including motors and plus-end tracking proteins (27,32,34,43,49). Specific PTMs are enriched on microtubules in restricted subcellular areas, suggesting that these mechanisms act as marks that locally adapt the microtubule polymer for specific functions (reviewed in reference 58). How the spatially restricted modified microtubules are generated within the cell is not well understood.Glutamylated microtubules are generated by the sequential addition of multiple glutamates to the ␥-carboxyl group of specific glutamic acids of the primary sequence of the C-terminal tail (CTT) domain of ␣-and ␤-tubulin (14). This reversible PTM creates glutamyl side chains of variable length and is enriched on microtubules in cilia, centrioles/basal bodies, the mitotic spindle, microtubules of nerve projections, and pellicular arrays in protists (2,5,6,31,35,45,63). Recently, we have identified tubulin glutamylases as proteins with a tubulin tyrosine ligase homology (28). The identification of forward enzymes for...

Two types of polymeric post-translational modifications of ␣/␤-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. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the ␣-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between ␣-and ␤-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.Microtubules are subject to a set of post-translational modifications (PTMs) 1 whose significance has emerged only recently (1-4). Among PTMs, two polymeric modifications, glutamylation and glycylation, substantially increase the heterogeneity of the ␣/␤-tubulin heterodimer. These tubulin modifications, referred to as polyglutamylation and polyglycylation, correspond to the addition of a peptide polymer consisting of several glutamates (5) or glycines (6) onto the ␥-carboxyl group of a glutamate of the primary sequence of tubulin. These two PTMs will be referred to as "polymodifications" throughout this report. Polymodifications generate peptide branches of variable lengths distributed on several glutamate acceptor sites in the C-terminal tails of ␣-and ␤-tubulin (7-9). Both polymodification types are enriched in flagella and cilia of protists and metazoan cells (4) and were implicated in axoneme motility (10, 11). Whereas polyglycylation is restricted to axonemes in the ciliated and flagellated metazoan cells, polyglutamylation occurs in both axonemes and basal bodies (12-15). In ciliates, both polymodification types are not only present in cilia and basal bodies (15-18), but also occur on the more dynamic intracytoplasmic microtubules (17,19,20). Ciliates assemble up to 17 types of distinct microtubular arrays in a single cell (19,(21)(22)(23). In these highly differentiated cells, the microtubular networks are involved in nuclear divisions, intracellular transport, organelle positioning, and are associated with specialized organelles that function in osmotic regulation, feeding, excretion, and locomotion. The ␣-and ␤-tubulins of ciliates are biochemically heterogeneous (17,20,24), suggesting that structural differences amon...

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono-and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the ␣-and ␤-tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

INTRODUCTIONParamecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.