COLD-ADAPTED MICROTUBULES - CHARACTERIZATION OF TUBULIN POSTTRANSLATIONAL MODIFICATIONS IN THE ANTARCTIC CILIATE EUPLOTES FOCARDII

In cold poikilotherm organisms, microtubule assembly is promoted at te mperatures below 4 degrees C and cold-induced depolymerization is prev ented. On the basis of the results of investigations on cold-adapted f ishes, the property of cold adaptation is ascribed to intrinsic charac teristics of the tubulins. To fully understand cold adaptation, we stu died the tubulins of Euplotes focardii, an Antarctic ciliated protozoa n adapted to temperatures ranging from -2 to +4 degrees C. In this org anism, we had previously sequenced one beta-tubulin gene and, then ide ntified three other genes (denoted as beta-T1, beta-T2, beta-T3 and be ta-T4). Here we report that the amino acid sequence of the carboxy-ter minal domain predicted from the beta-T3 gene (apparently the most expr essed of the gene family) contains six modifications (five substitutio ns and one insertion) of conserved residues, unique with respect to al l the other known beta-tubulin sequences. These modifications can chan ge the structural conformation of the carboxy-terminal domain. Further more, in the variable terminal end of that domain, a consensus sequenc e for a phosphorylation site is present, and the residue Glu-438, the most frequent site for polyglutamylation in beta-tubulin, is substitut ed by Asp. Starting from these observations, we showed that in E. foca rdii only alpha-tubulin is polyglutamylated, while beta-tubulin underg oes phosphorylation. Polyglutamylated microtubules appear to colocaliz e with cilia and microtubular bundles, all structures in which microtu bules undergo a sliding process. This finding supports the idea that a lpha-tubulin polyglutamylation is involved in the interaction between tubulin and motor microtubule-associated proteins. Phosphorylation, us ually 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 polymerizatio n and depolymerization at low temperatures. (C) 1997 Wiley-Liss, Inc.