Post-Translational Modifications to Toxoplasma gondii α- and β-Tubulins Include Novel C-Terminal Methylation

Abstract

Toxoplasma gondii is an apicomplexan of both medical and veterinary importance which is classified as an NIH Category B priority pathogen. It is best known for its ability to cause congenital infection in immune competent hosts and encephalitis in immune compromised hosts. The highly stable and specialized microtubule-based cytoskeleton participates in the invasion process. The genome encodes three isoforms of both α-and β-tubulin and we show that the tubulin is extensively altered by specific post-translational modifications (PTMs) in this paper. T. gondii tubulin PTMs were analyzed by mass spectrometry and immunolabeling using specific antibodies. The PTMs identified on α-tubulin included acetylation of Lys40, removal of the last C-terminal amino acid residue Tyr453 (detyrosinated tubulin) and truncation of the last five amino acid residues. Polyglutamylation was detected on both α-and β-tubulins. An antibody directed against mammalian α-tubulin lacking the last two C-terminal residues (Δ2-tubulin) labeled the apical region of this parasite. Detyrosinated tubulin was diffusely present in subpellicular microtubules and displayed an apparent accumulation at the basal end. Methylation, a PTM not previously described on tubulin, was also detected. Methylated tubulins were not detected in the host cells, human foreskin fibroblasts, suggesting that this may be a modification specific to the Apicomplexa.

Post-translational modifications of microtubules

“…Organisms that lack tubulin glycylation could use other means to inhibit tubulin glutamylation, including tubulin deglutamylation (see below) or other competing PTMs. In Toxoplasma gondii, a potentially polymodifiable residue of -tubulin, E434, undergoes carboxyl methylation (Xiao et al, 2010), a PTM of unknown effect, that could also compete with tubulin glutamylation (Plessmann et al, 2004). Biochemical studies have detected activities that shorten polymodification side chains: deglutamylation (Audebert et al, 1993) and deglycylation (Bre et al, 1998).…”

Lysine Acetylation Is Widespread on Proteins of Diverse Function and Localization in the Protozoan Parasite Toxoplasma gondii

“…Lysine acetylation occurs on Toxoplasma histones, but studies suggest that lysine acetylation may be more extensive. A proteomics study mapping the posttranslational modifications of Toxoplasma tubulins revealed acetylation of lysine 40 (K40) on ␣-tubulin (46), which is a conserved modification in most eukaryotes excluding yeasts (40). We have also noted that several Toxoplasma lysine acetyltransferases (KATs) are predominantly cytoplasmic, suggesting that they may have substrates in the parasite cytosol.…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…Moreover, specific isotypes can be required for specialized structures, such as the flagellar axoneme (20). The Toxoplasma genome contains genes for three ␣ and three ␤ isotypes (21,22). The Toxoplasma ␣1 isotype is abundantly represented in tachyzoite and oocyst proteomes and is mutated in all dinitroaniline-resistant lines described to date (23)(24)(25).…”

“…Toxoplasma ␣1-tubulin can be modified by acetylation of lysine 40 and detyrosination (removal of the carboxy-terminal tyrosine 453) (22). Notably, only ␣1-tubulin is susceptible to both modifications: ␣2-tubulin lacks a lysine 40 and ends in a valine, while ␣3-tubulin ends with an asparagine.…”