Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

Abstract

In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of ␣-and ␤-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (51). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the ␥-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of ␣-and ␤-tubulin (14, 36). Glutamylated microtubules are abundant in projections of neurons (14), axonemes (8,15,17), and centrioles/basal bodies (5, 31) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (1, 5). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (22,50,53). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (20). A loss of PGs1, a protein associated with TTLL1 E-ligase (22, 37), disorganizes sperm axonemes in the mouse (11), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (33). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubuleassociated proteins (MAPs) (6,7,19,27).The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (8, 53). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (53). In Tetrahymena, Ttll6Ap is a ␤-tubulin-preferring E-ligase (22), wi...

Unique Post-Translational Modifications in Specialized Microtubule Architecture

“…Interestingly, TTLL3-deficient Tetrahymena shows a counter-increase in polyglutamylation along with a decrease in tubulin polyglycylation (Wloga et al, 2009b), which is consistent with the concept of cross-talk of PTMs in α-and β-tubulin C-termini (Redeker et al, 2005). The increase in polyglutamylation could explain the cause of abnormal cilia formation, as over-polyglutamylation, manipulated by the overexpression of the polyglutamylase TTLL6, destabilizes axonemal microtubules (Wloga et al, 2009a). These findings can provide a cue for understanding the molecular machinery whereby tubulin polyglycylation performs its function.…”

Writing and Reading the Tubulin Code

“…TTLL1, -4, -5, -6, -7, -9, -11, and -13 are glutamylases (3,6,39,77,78), whereas TTLL3, -8, and -10 are glycylases ((4, 7, 79); reviewed in Ref. 43).…”

Tubulin Tyrosine Ligase-like Genes ttll3 and ttll6 Maintain Zebrafish Cilia Structure and Motility

“…Drosophila mutants lacking the ␤-tubulin C terminus fail to assemble sperm axonemes, whereas cytoplasmic microtubules are not affected (17, 18). Axonemal B-subfiber defects and reduced cilia beat amplitude are also observed in the zebrafish fleer mutant, which lacks a TPR protein required for cilia polyglutamylation (19), as well as in Tetrahymena overexpressing the tubulin glutamylase, TTLL6Ap (20,21). Evidence for a role for tubulin glycylation in cilia elongation in zebrafish has recently been reported (22).…”