Genetic Evidence for Interaction between η- and β-Tubulins

Abstract

The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, ␣ to , characterized in P. tetraurelia. In order to analyze -tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the ␤-PT2 gene coding for a ␤-tubulin, while the mutation noc r 1 conferring nocodazole resistance and localized in another ␤-tubulin gene, ␤-PT3, was shown to enhance the mutant phenotype. The interaction between -tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that -tubulin, like ␥-and ␦-tubulins, might be a microtubule minus-end capping molecule. The likely function of -tubulin as part of a complex specifically involved in basal body biogenesis is discussed.The highly conserved ␣-and ␤-tubulins constitute the building blocks of microtubule arrays, and members of a third tubulin subfamily, ␥-tubulin, involved in the polymerization of ␣/␤ dimers into microtubules, have been identified in all eukaryotes. In addition to these three ubiquitous subfamilies, several new members of the tubulin family have been discovered in the last few years: delta (14), epsilon (5), zeta (39), eta (30), and iota and theta (P. Dupuis-Williams, unpublished data; GenBank/EMBL/DDBJ accession numbers AJ427481 and AJ427480). Interestingly, while some of the new tubulins were identified through genome survey, two of them, ␦ and , were identified by complementation cloning of mutations affecting basal body assembly/duplication, providing a first hint as to their function. In particular, a role of ␦-tubulin in the C-tubule assembly of basal bodies, deduced from the properties of the UNI3 mutant of Chlamydomonas reinhardtii (14), was confirmed in Paramecium tetraurelia by the effect of ␦-tubulin gene silencing (16).A function of ε-tubulin in centriole/basal body assembly was recently demonstrated (4,11,13) and is consistent with the fact that ε-tubulin, like ␦-tubulin, is found in widely distant organisms-mammals, green algae, protozoans-but is absent from the completely sequenced genomes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans, which do not possess such organelles or possess highly divergent ones. As for -, -, -, and -tubulins, they do not seem so far to have metazoan homologues, and their functions might be restricted to ciliates and flagellates. However, since -tubulin is involved in basal body duplications but it has no homologue in other organisms, its function in basal body duplication might be accompli...

Paramecium tetraurelia basal body structure

“…In addition, basal body duplication is impaired after ε-tubulin depletion [ 20 ]. A mutation found in sm19 , encoding the rare eta now called ζ [ 21 ], was reported to inhibit basal body duplication and to induce the delocalization of γ-tubulin [ 22 , 23 ].…”

“…The mutations in the gene encoding this protein caused an inhibition of basal body duplication [ 42 ]. Further physiological and genetical studies indicate an interaction with microtubules [ 22 ]. In a recent functional study of ζ-tubulin in Xenopus [ 21 ], no evident basal body defect was observed, leading to the conclusion that ζ-tubulin is involved in basal body orientation and distribution and might function by interacting with other tubulins.…”

Basal Body Assembly in Ciliates: The Power of Numbers

“…Finally, forward genetic strategies identified a Paramecium ‐specific tubulin superfamily member, η‐tubulin that was originally found as a conditional mutation ( sm19 ) that, at restrictive temperature, inhibited basal body duplication (85). η‐Tubulin is a divergent tubulin that genetically interacts with β‐tubulin in a manner suggesting that the proteins directly interact (86). Furthermore, γ‐tubulin is mislocalized in η‐tubulin mutants (85).…”

Polarity in Ciliate Models: From Cilia to Cell Architecture

“…For example, in T. thermophila GFP-BTU2 is found in somatic cilia and BBs, whereas GFP-BLT1 and GFP-BLT4 are observed in macronucleus MT arrays and in the mitotic spindle of the micronucleus (Pucciarelli et al, 2012). In Paramecium the tubulin gene family possesses two α- and three β-tubulin genes (Dupuis, 1992; Dupuis-Williams et al, 1996; Ruiz et al, 2004).…”