Glutamylation on α-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 ␣-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...

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

“…7, A and B). This antiserum recognizes longer chains of polyE (45,46). These results indicate that the loss of CCP1 results in hyperglutamylation of ␤-tubulin as well as ␣-tubulin.…”

Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases

“…Furthermore, the enzymes that catalyze the glycylation on tubulin and on other substrate proteins have been identified as members of the tubulin tyrosine ligase-like (TTLL) 2 family, which also includes other amino acids ligases such as the tubulin tyrosine ligase (TTL) (11) and polyglutamylases, which add glutamic acid instead of glycines (12)(13)(14). Glycylases can be classified as: primases, which add the first glycine, like TTLL3s of vertebrates and Tetrahymena thermophila and the mammalian TTLL8 (9, 10, 15); elongases, which are only able to elongate the polyglycine chain, like mammalian TTLL10, with the exception of the non-functional human TTLL10 (9, 10); bifunctional initiating/elongating enzymes, like Drosophila melanogaster dmTTLL3A and dmTTLL3B (10).…”

“…Polyglycylation has been related to the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of the protein inside the nuclei. In fact, in G. duodenalis parasites expressing the g14-3-3 mutant E246A, which cannot be polyglycylated, the protein localizes in the nuclei throughout the parasite life cycle, resulting in a faster differentiation of the trophozoite into the cyst stage once the process has been induced (3, 4).Furthermore, the enzymes that catalyze the glycylation on tubulin and on other substrate proteins have been identified as members of the tubulin tyrosine ligase-like (TTLL) 2 family, which also includes other amino acids ligases such as the tubulin tyrosine ligase (TTL) (11) and polyglutamylases, which add glutamic acid instead of glycines (12)(13)(14). Glycylases can be classified as: primases, which add the first glycine, like TTLL3s of vertebrates and Tetrahymena thermophila and the mammalian TTLL8 (9, 10, 15); elongases, which are only able to elongate the polyglycine chain, like mammalian TTLL10, with the exception of the non-functional human TTLL10 (9, 10); bifunctional initiating/elongating enzymes, like Drosophila melanogaster dmTTLL3A and dmTTLL3B (10).…”

Unique Post-Translational Modifications in Specialized Microtubule Architecture

“…The first identified TTLL1 is a subunit of the polyglutamylase complex purified from mouse brain (Janke et al, 2005). Further studies demonstrate that TTLL4, 5, 6, 7, 9, 11, and 13 have tubulin polyglutamylase activity (Ikegami et al, 2006;van Dijk et al, 2007;Wloga et al, 2008;Mukai et al, 2009). Currently, the glutamate-removing enzyme, deglutamylase or glutamatase remains to be identified.…”