Research Article

Tubulin Polyglutamylase Enzymes Are Members of the TTL Domain Protein Family

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Science  17 Jun 2005:
Vol. 308, Issue 5729, pp. 1758-1762
DOI: 10.1126/science.1113010


Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase–like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Polyglutamylation is an uncommon type of post-translational modification that adds multiple glutamic acids to a γ-carboxyl group of a glutamate residue of target proteins, including tubulin and nucleosome assembly proteins NAP1 and NAP2 (14). The resulting polyglutamate side chains are of variable length, allowing for a graded regulation of protein-protein interactions. Polyglutamylation regulates the binding of neuronal microtubule (MT)–associated proteins as a function of the length of the polyglutamate chain, which suggests that the modification is important for the organization of the neuronal MT network (5). Tubulin polyglutamylation may also play a role in centriole stability (6), axoneme motility (7, 8), and mitosis (9, 10).

Identification of mouse brain polyglutamylase. Monoclonal antibody (mAb) 206, raised against a partially purified brain tubulin polyglutamylase fraction, immunoprecipitates the enzyme complex, including the PGs1 (polyglutamylase subunit 1) protein p32 (11). We used two-dimensional (2D) gel electrophoresis followed by nano-LC-MS-MS (liquid chromatography–mass spectrometry) to identify four additional protein components of the same complex: p24, p33, p49, and p79 (Fig. 1A) (table S1). Polyclonal antibodies were raised against recombinant proteins or peptides of p24, p32, p33, p49, and p79 and used for coimmunoprecipitation analyses; mAb 206, polyclonal antibody to p32 (L83), and polyclonal antibody to p79 (L80) precipitated ≥80% of the polyglutamylase activity (Fig. 1B) along with all five proteins (Fig. 1C). These five proteins also consistently copurified with the polyglutamylase activity during several purification steps (fig. S1); therefore, p33, p49, p79, and p24 were named PGs2, PGs3, PGs4, and PGs5, respectively. Additional proteins were found in the mAb 206–immunoprecipitated fraction, including Arp1 and CF Im25 (table S1), but these did not consistently copurify with the enzyme activity.

Fig. 1.

Characterization of the neuronal polyglutamylase. (A) Coomassie Brilliant Blue–stained nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS–polyacrylamide gel electrophoresis (PAGE) 2D gel of a purified polyglutamylase fraction, immunoprecipitated from 200 3-day-old mouse brains with mAb 206. All numbered protein spots were identified by nano-LC-MS-MS (table S1). HC and LC mark the positions of heavy and light chains of mAb 206. (B) Immunoprecipitations with mAb 206, L83 [antibody to p32 (11)], L80 (antibody to p79), and DM1A (α-tubulin mAb) used as a control. Equal proportions of input and supernatants were assayed for polyglutamylase activity. (C) Input, supernatants, and beads (equal proportions) were analyzed by immunoblotting with antibodies L26, L80, L83, L90, and L91 and antibody to Arp1. The polyglutamylase activity and all five polyglutamylase complex subunits were strongly depleted from the supernatants. The proteins were quantitatively recovered with the beads of mAb 206, L83, and L80, but not of DM1A. Arp1 did not quantitatively copurify with the enzyme.

The apparent size of the neuronal polyglutamylase complex, 360 kD (12), is greater than the theoretical sum of the predicted molecular masses of all five subunits (217 kD), which implies that the complex contains multiple copies of one or more subunits. Because some of the components appear on 2D gels as multiple spots at different isoelectric points, they may themselves be subject to charge-altering post-translational modifications (Fig. 1A). Indeed, PGs1 was phosphorylated on Ser279 (13).

PGs1 (a product of the mouse gene GTRGEO22) is required for sperm axoneme assembly and normal animal behavior (14) and may act in the intracellular targeting of the polyglutamylase complex (11). PGs3 is an ortholog of the human TTLL1 protein (15). The amino acid sequence of TTLL1 exhibits 17% identity with tubulin tyrosine ligase (TTL), which catalyzes the addition of tyrosine to the C-terminal glutamate of detyrosinated α-tubulin (16). Despite obvious differences, both polyglutamylation and tyrosination reactions involve an amino acid addition to a glutamate residue through the formation of an amide bond. Thus, we examined the possibility that TTLL1 is the catalytic subunit of neural tubulin polyglutamylase.

A structural model for TTLL1. The amino acid sequence of TTLL1 (PGs3) contains three conserved motifs that correspond to the adenosine triphosphate (ATP)/Mg2+-binding site typical of enzymes with a carboxylateamine/thiol ligase activity, such as glutathione synthetase (17). Although the overall sequence similarity between TTLL1 and the known carboxylate-amine/thiol ligase enzymes is low, we could align the ATP-binding regions as well as all major parts of TTLL1. Using glutathione synthetase from Escherichia coli as a template, we obtained a structural model of TTLL1 by homology-based modeling (Fig. 2, A and B). Docking of ATP and Mg2+ into the model supports the localization of the ATP/Mg2+-binding site (Fig. 2, B and C) (fig. S2). We were also able to fit a peptide with a polyglutamate side chain into the active site, which located a putative binding site for free glutamate (Fig. 2C). Thus, it is likely that TTLL1 protein is the catalytic subunit of neural tubulin polyglutamylase.

Fig. 2.

A structural model of TTLL1. Cyan arrows represent β sheets, α helices are magenta, and loops are blue. (A) Crystal structure of glutathione synthetase from E. coli (PDB code 1GSA) in a complex with adenosine diphosphate (ADP), glutathione, Mg2+, and sulfate (27). (B) A model of mouse TTLL1 (PGs3) in a complex with ATP, Mg2+, and a protein substrate. The regions of TTLL1 that could not be modeled are drawn as thin lines. (C) A close-up view of the active center of TTLL1. The protein substrate is a three-residue peptide with a central modified glutamate (backbone in medium green). The flanking amino acids are drawn only with Cβ atoms for clarity. The glutamate side chain contains two additional glutamate residues (Glu 1, Glu 2). The putative site for the next glutamate residue to be added to the side chain is indicated (Glu 3 site). All residues of the active site that are conserved with other ATP-dependent carboxylateamine ligases are shown in dark green (see fig. S2). Some positively charged amino acid residues (Lys13, Lys142, and Lys215 in light gray) that are specific to TTLL1 and close to the active site might be important for substrate binding. The proximity of the carboxyl group of Glu 2 and the phosphate of ATP could catalyze the formation of an acylphosphate intermediate (broken line). Oxygen, nitrogen, phosphate, and magnesium atoms are in red, blue, magenta, and orange, respectively.

Ttll1p is associated with α-tubulin polyglutamylase activity in vivo. When expressed in bacteria or in various cell lines as well as in several heterologous systems, the murine TTLL1 had a strong tendency to precipitate and did not show polyglutamylase activity in vitro. We used a homologous protein expression system based on the ciliated protist Tetrahymena thermophila to assess the role of TTLL1-related proteins in polyglutamylation. Tetrahymena has a complex cytoskeleton with a large number of distinct types of MTs (18). Most types of MTs in Tetrahymena are monoglutamylated, although a small subset—including MTs of the basal bodies (BBs), cilia, contractile vacuole pore (CVP), and oral deep fiber (DF)—have side chains composed of two or more glutamates (19). Using the recently sequenced macronuclear genome of Tetrahymena (20), we identified the likely TTLL1 ortholog Ttll1p (54% amino acid sequence identity with TTLL1). Ttll1p with an N-terminal green fluorescent protein (GFP) was strongly overexpressed by means of a cadmium-inducible gene promoter (21). Ttll1p-GFP localized to a subset of polyglutamylated MT organelles including BBs, CVPs, and DF (Fig. 3A), but no increase in the level of MT polyglutamylation over normal levels was detected (Fig. 3, B, C, and E) and the phenotype appeared normal. Similar results were obtained for the hemagglutinin epitope C-terminally tagged protein (22). No change in tubulin polyglutamylation activity in vitro was detected in cell extracts despite the strong accumulation of Ttll1p-GFP (Fig. 3F). However, a polyglutamylase activity directed mostly toward α-tubulin was coimmunoprecipitated with antibody to GFP from extracts of overexpressing cells (Fig. 3, G and H). The overexpressed protein apparently could replace a part of the endogenous Ttll1p but could not function alone. On the basis of the data obtained for the murine homolog, it is likely that Ttll1p also acts in a complex and that other subunits are limiting.

Fig. 3.

Ttll1p of Tetrahymena is associated with tubulin glutamylation. (A) A cell expressing Ttll1p-GFP, labeled by immunofluorescence using antibody to GFP. (B to D) Polyglutamylated MTs labeled by mAb ID5 in a cell overproducing Ttll1p-GFP (B), a wild-type cell (C), and a TTLL1-null cell (D). Note a strong reduction in the labeling of rows of basal bodies in (D) relative to (C). Abbreviations: oa, oral apparatus; noa, new oral apparatus before cell division; df, deep fiber; bb, basal bodies. (E) Immunoblotting studies on total Tetrahymena cell extracts before (–) and after (+) 3 hours of cadmium induction of Ttll1p-GFP, analyzed with antibodies to GFP and to polyglutamylation (mAb GT335 and antibody polyE). An arrowhead points to Ttll1p-GFP; an asterisk marks the comigrating α- and β-tubulin bands. (F) Soluble (S) and cytoskeletal (C) fractions from cells overproducing Ttll1p-GFP (+) or noninduced controls (–) were analyzed for tubulin polyglutamylase activity in vitro. (G and H) Soluble fractions of cells overproducing Ttll1p-GFP were subjected to immunoprecipitation with antibody to GFP. The input (In), unbound (Un), and bound (Bo) fractions were analyzed by immunoblotting with antibody to GFP (arrowhead TG points to Ttll1p-GFP, HC to the antibody heavy chain) (G) and assayed for glutamylase activity in vitro (H). Equal volumes of input, unbound, and bead fractions were analyzed by immunoblotting; a volume of bead fraction 20 times this size was assayed for glutamylase activity.

We also used gene disruption to construct cells completely lacking the TTLL1 gene. The TTLL1-null cells had a normal phenotype but showed a strong reduction in tubulin polyglutamylation in the BBs (Fig. 3, C and D). This result confirms that Ttll1p is involved in polyglutamylation but also suggests that there are additional polyglutamylase activities in this organism that do not require Ttll1p.

The TTLL family. TTLL1 and TTL are members of a large family of conserved eukaryotic proteins with a TTL homology domain, which raises the possibility that other members of this family are also involved in polyglutamylation or other types of posttranslational amino acid ligations. Phylogenetic analyses showed that TTLLs of diverse eukaryotes belong to several conserved subtypes (Fig. 4) (figs. S3 and S4). We used the HsTTLL1 sequence (15) as a template for tBLASTn searches to identify all TTLL loci of Tetrahymena and several other model eukaryotes. Phylogenetic analyses revealed 10 clades of TTLLs, eight of which contain predicted mammalian proteins. Tetrahymena has one to seven sequences in most groups, as well as one clade of 20 ciliate-specific TTLLs. Among the genomes surveyed, only Trypanosoma has a close homolog of the mammalian TTL, which may be why an enzymatic activity of TTL was not detected in invertebrates (23) and Tetrahymena (24).

Fig. 4.

An evolutionary tree of TTL domain proteins, based on the neighbor-joining method. Numbers correspond to bootstrap values for 100 repeats. Colored dots represent predicted TTL domain sequences in several genomes analyzed. Gray boxes denote clades discussed in the text. The yeast sequence was used as an outgroup. See figs. S3 and S4 for complete data.

Tetrahymena Ttll6Ap is a β-tubulin–preferring polyglutamylase. The Tetrahymena TTLL6A sequence belongs to a clade related to TTLL1 (Fig. 4) (fig. S3). Over-produced Ttll6Ap-GFP localized mainly to cilia, with a small amount associated with BBs and cell body MTs (Fig. 5A). Overproduction of Ttll6Ap-GFP led to a strong increase in polyglutamylation in cilia and on cell body MTs (Fig. 5C; compare with Fig. 3C). A strong increase in tubulin polyglutamylation (but not in polyglycylation) was also detected in whole cells by immunoblotting (Fig. 5F).

Fig. 5.

Ttll6Ap-GFP is associated with strong tubulin polyglutamylation in vivo and in vitro. (A to D) GFP fluorescence [(A) and (B)] and immunofluorescence images with mAb ID5 [(C) and (D)] of cells overproducing Ttll6Ap-GFP [(A) and (C)] or Ttll6ApΔ710-GFP [(B) and (D)]. The staining detected in (A) and (B) colocalized with MTs visualized by mAb 12G10 to α-tubulin (22). Strong polyglutamylation appears in the cell body in cells overproducing forms of Ttll6Ap, whereas polyglutamylation is restricted to cilia and basal bodies in control cells (compare with the wild-type cell in Fig. 3C). Abbreviations: c, cilia; oc, oral cilia; oa, oral apparatus; bb, basal bodies; sc, subcortical MTs; ic, intracytoplasmic MTs. (E) Tubulin polyglutamylase assays using soluble (S) and cytoskeletal (C) fractions from Ttll6ApΔ710-GFP cells grown in the absence (–) and presence (+) of cadmium for 3 hours. (F) Immunoblots of total cells or cytoskeletons of the following strains after 3 hours of cadmium treatment: wild type (WT), cells overproducing GFP (GFP), Ttll6ApΔ710-E422G-GFP (Δ710-G), Ttll6ApΔ710-GFP (Δ710), and Ttll6Ap-GFP (Ttll6Ap). Antibodies used are shown at the right. (G to I) Cells overproducing an enzymatically active Ttll6Ap and not an ATPase-deficient form fail to multiply and undergo ciliary paralysis. Growth curves on SPP medium are shown for strains expressing Ttll6Ap-GFP (G), Ttll6ApΔ710-GFP (H), or Ttll6ApΔ710-E422G-GFP (H) with or without cadmium induction. Note that only the ATPase-capable proteins reduce the growth rate. (I) Percentage of motile cells in several overproducing strains. After 3 hours of cadmium induction, 100 to 200 cells were scored for vigorous motility. Either extremely sluggish (showing rotations but no directional movement) or completely paralyzed cells were counted as nonmotile. Data in (G) to (I) are mean values from three independent experiments.

A truncated variant lacking the 286 C-terminal amino acids, Ttll6ApΔ710-GFP, localized predominantly to the cell body with a strong preference for subcortical MTs that extend from the apical end of the cell and run below the BBs (Fig. 5B). Consistently, overexpression of Ttll6ApΔ710-GFP led to a strong increase in polyglutamylation on MTs in the cell body and to a much lesser extent in cilia (Fig. 5D). Thus, the 286 C-terminal amino acids of Ttll6Ap are required for preferential targeting to cilia.

We used the truncated version of Ttll6Ap for biochemical studies because the proportion of this variant in the soluble/cytosolic pool is increased relative to the full-length protein. Extracts of cells overexpressing Ttll6ApΔ710-GFP showed a 100-fold increase in polyglutamylase activity in vitro for β-tubulin and a 10-fold increase for α-tubulin relative to noninduced cells (Fig. 5E). This activity copurified with Ttll6ApΔ710-GFP protein under all conditions tested (fig. S5, A to C). No in vitro activity toward NAP proteins was detected. Thus, Ttll6Ap is a tubulin polyglutamylase displaying a strong preference for the β-tubulin subunit.

Increased polyglutamylation affects cell growth and motility. Tetrahymena cells overexpressing Ttll6Ap-GFP ceased to multiply within a few hours after cadmium induction (Fig. 5G), and most had paralyzed cilia (Fig. 5I), indicating that excessive polyglutamylation inhibits cell proliferation and ciliary dynein-based motility. Cells overexpressing the truncated protein also ceased to proliferate, but the effect on ciliary motility was much weaker, in accordance with the altered protein localization pattern (Fig. 5, H and I). These effects did not occur when a mutation in the predicted ATP binding site of the TTL homology domain (Glu422 → Gly, E422G) was introduced (fig. S2). Relative to the ATPase-active protein, the inactive variant, Ttll6ApΔ710-E422G-GFP, was expressed at a similar level (Fig. 5F) and localized to the same types of MT organelles (22). However, no increase in tubulin polyglutamylation was observed (Fig. 5F), nor were alterations of cell growth or motility observed (Fig. 5, H and I); these findings confirm that excessive polyglutamylation, not the overproduced protein, was responsible for the observed phenotypes.

Conclusion. The simplest interpretation of our data is that TTLL1/Ttll1p and Ttll6Ap are two types of tubulin polyglutamylase catalytic components with distinct tubulin subunit preferences. The neuronal TTLL1 as well as Ttll1p have a preference for α-tubulin, whereas Ttll6Ap preferentially polyglutamylates β-tubulin. Unlike Ttll6Ap, Ttll1p (and its murine ortholog) did not increase polyglutamylation in vitro and in vivo upon overproduction. However, TTLL1 exists in a protein complex, and additional subunits may be required for its activity and could be limiting in vivo. Indeed, immunoprecipitation of Ttll1p-GFP from overproducing Tetrahymena cells led to a recovery of polyglutamylase activity.

Ttll6Ap is a much larger protein (116 kD) than TTLL1 (49 kD) and Ttll1p (42 kD), and it may contain all properties required for autonomous polyglutamylase activity. The four non-catalytic subunits identified in the neuronal TTLL1 complex may be involved in tubulin substrate recognition, regulation of enzymatic activity, or subcellular localization, as has been suggested for PGs1 (11). It is likely that Ttll1p is also in a complex, as is the murine homolog. Except for PGs4, we could not identify homologs of the other subunits of the neural complex (PGs1, PGs2, PGs5) outside of vertebrates, including Tetrahymena; therefore, variations in the composition of noncatalytic subunits likely occur across phyla. The unusually large number of TTLL genes in Tetrahymena and the lack of a detectable loss-of-function phenotype for TTLL1 suggest functional redundancy. In contrast, a mutation in the PGs1 component of the murine TTLL1 complex led to defective sperm axonemes and changes in animal behavior (14). In Caenorhabditis elegans, RNA interference (RNAi) depletion of C55A6.2 (a TTLL5 type) causes embryonic lethality and sterility (25). Depletion of TTLL1 mRNA in PC12-E2 cells inhibited neurite outgrowth, suggesting an essential function in neurogenesis (22).

The phylogenetic association of TTLL1, TTLL9, TTLL4, TTLL6, TTLL5, and TTLL15 protein types (86% bootstrap value; Fig. 4) suggests that these protein types are all involved in glutamylation of tubulin or possibly of other proteins such as NAPs (4). Other members of the TTLL family may catalyze different types of posttranslational addition of an amino acid, such as polyglycylation (26).

Supporting Online Material

Materials and Methods

Figs. S1 to S5

Table S1


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