Cold-adapted microtubules: characterization of tubulin posttranslational modifications in the Antarctic ciliate...

In cold poikilotherm organisms, microtubule assembly is promoted at temperatures below 4 degrees C and cold-induced depolymerization is prevented. On the basis of the results of investigations on cold-adapted fishes, the property of cold adaptation is ascribed to intrinsic characteristics of the tubulins. To fully understand cold adaptation, we studied the tubulins of Euplotes focardii, an Antarctic ciliated protozoan adapted to temperatures ranging from -2 to +4 degrees C. In this organism, we had previously sequenced one beta-tubulin gene and, then identified three other genes (denoted as beta-T1, beta-T2, beta-T3 and beta-T4). Here we report that the amino acid sequence of the carboxy-terminal domain predicted from the beta-T3 gene (apparently the most expressed of the gene family) contains six modifications (five substitutions and one insertion) of conserved residues, unique with respect to all the other known beta-tubulin sequences. These modifications can change the structural conformation of the carboxy-terminal domain. Furthermore, in the variable terminal end of that domain, a consensus sequence for a phosphorylation site is present, and the residue Glu-438, the most frequent site for polyglutamylation in beta-tubulin, is substituted by Asp. Starting from these observations, we showed that in E. focardii only alpha-tubulin is polyglutamylated, while beta-tubulin undergoes phosphorylation. Polyglutamylated microtubules appear to colocalize with cilia and microtubular bundles, all structures in which microtubules undergo a sliding process. This finding supports the idea that alpha-tubulin polyglutamylation is involved in the interaction between tubulin and motor microtubule-associated proteins. Phosphorylation, usually a rare posttranslational modification of beta-tubulin, which is found extensively distributed in the beta-tubulin of this cold-adapted organism, may play a determinant role in the dynamic of polymerization and depolymerization at low temperatures.