Multiple phosphorylation sites on γ-tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena.

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.