Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-spe cific antibodies. Three and five polyglycylation sites were identified at g lutamic acids near the COOH termini of alpha- and beta-tubulin, respectivel y. Mutants lacking all polyglycylation sites on alpha-tubulin have normal p henotype, whereas similar sites on beta-tubulin are essential. A viable mut ant with three mutated sites in beta-tubulin showed reduced tubulin glycyla tion, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubu- lin gene whose COOH terminus wa s replaced by the wild-type COOH terminus of beta-tubulin. In this double m utant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-ty pe cells. Thug the essential function of polyglycylation of the COOH termin us of beta-tubulin can be transferred to alpha-tubulin, indicating it is th e total amount of polyglycylation on both alpha- and P-tubulin that is esse ntial for survival.