Mutations of Tubulin Glycylation Sites Reveal Cross-talk between the C Termini of α- and β-Tubulin and Affect the Ciliary Matrix in Tetrahymena

Abstract

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...

Crystal structure of tubulin tyrosine ligase-like 3 reveals essential architectural elements unique to tubulin monoglycylases

“…Unexpectedly, we find that at high levels of β-tubulin tail glutamylation, α-tail glycylation by TTLL3 is enhanced. This finding is consistent with prior in vivo studies in Tetrahymena showing that removal of glycylation/glutamylation sites in the β-tail enhances α-tail glycylation (26). Although the functional significance of this observation remains to be explored, our study provides a blueprint for investigating how tubulin modification patterns are generated in vivo through the intersection of activities of multiple TTLL enzymes.…”

“…Here we focus on the mechanistic dissection of TTLL3 and show that it strictly initiates glycyl chains on the β-tubulin tail using a positional hierarchy. Moreover, we find that TTLL3 competes with the glutamylase TTLL7 for overlapping modification sites on the β-tail, providing a molecular basis for the anticorrelation between glutamylation and glycylation observed in vivo (12,25,26). Consistent with this competition for modification sites, TTLL3 activity is reduced on glutamylated versus unmodified microtubules.…”

“…Therefore, TTLL3 and TTLL8 likely encounter tubulin that is already glutamylated. We demonstrate with purified components that the glycylase TTLL3 and glutamylase TTLL7 compete for overlapping initiating sites on the β-tail, providing a molecular basis for the observed negative correlation between glutamylation and glycylation in vivo (13,25,26,32). Consistent with this competition, TTLL3 activity is drastically reduced on microtubules with abundant glutamylation.…”

“…The TTLL3-modified sites we identify concentrate on the last five glutamates in βΙ, previously found to be glycylated in β-tubulin isolated from Paramecium tetraurelia (30) and Purpurus lividus (31). Competition for substrate between glutamylases and glycylases has been proposed as a regulatory mechanism (13,26,32) because inhibition or overexpression of glutamylases causes marked increases or decreases in glycylation and vice versa (13). To test this hypothesis, we used MS/MS to sequence the βI-tubulin tails from microtubules glutamylated by TTLL7, a β-tubulin-specific glutamylase that initiates and elongates glutamate chains (33).…”

Cell biology of embryonic migration

“…Some of these tubulin modifications have been associated with microtubule stability and found to serve as markers for cell polarization (Erck et al, 2005;Gundersen and Bulinski, 1986;Infante et al, 2000;Khawaja et al, 1988). Tubulin modifications by amino acids have also been implicated in modulation of microtubule functions in various types of motile cells (Ikegami et al, 2007;Raybin and Flavin, 1977a;b;Redeker et al, 2005).…”