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Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo.

ABSTRACT: The C termini of beta-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human betaI and betaIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from betaIII and 16 from betaI can assemble normally. Interestingly, removal of the next residue (Ala(428)) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human betaI and betaIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1-429 of human beta-tubulins make no contribution to microtubule assembly. Ala(428), in the C-terminal sequence motif N-QQYQDA(428), lies at the end of helix H12 of beta-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala(428) may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr(429) plays a more complex role. In the betaI isotype of tubulin, Thr(429) is not at all necessary for assembly; however, in the betaIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of betaIII as well as with the observation that evolutionary conservation is total for Ala(428) and frequent for Thr(429).

SUBMITTER: Joe PA

PROVIDER: S-EPMC3837401 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo.

Joe Patrick A PA Banerjee Asok A Ludueña Richard F RF

The Journal of biological chemistry 20081213 7

The C termini of beta-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human betaI and betaIII tubulin. Transient t ...[more]

PMID: 19074767

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Project description:BackgroundThe multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.Methodology/principal findingsWith respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.Conclusion/significanceWe conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

Dataset Information

Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo.

Publications

Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo.

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