This proposal is to study the significance of post- translational modifications of tubulin in the ciliate, Tetrahymena thermophila. The project is based upon a recent achievement by the principal investigator, while he was a postdoctoral fellow, that permits genetic transformation of Tetrahymena by homologous integration. Both macro- and micro-nuclear transformation are now possible, allowing for the study of phenotype following gene replacement. Tetrahymena possesses a single alpha and two beta tubulin genes. This proposal will examine the phenotype of alleles of alpha tubulin that have been engineered to block each of tubulin's known post-translational modifications. Additional work will seek currently unknown post- translational modifications (PTMs), and again, the phenotype of their elimination will be studied. The hypothesis to be tested is that PTMs are associated with and significant for tubulin differentiation. This idea is certainly plausible in Tetrahymena, given that the same alpha tubulin is employed for several distinct microtubule systems within the cell: cilia, cortical microtubules, the mitotic spindle, etc. Biochemistry will be carried out on ciliary alpha tubulins of Tetrahymena to identify additional PTMs that may be important for tubulin function in this organelle. It is proposed that this analysis in Tetrahymena will elucidate the role of PTMs in microtubules differentiation for other eukaryotic systems.
