Tubulin glycylation and glutamylation are two related post-translational modification typesbased on addition of either multiple glycines or glutamic acid residues to - and -tubulin. Theseso-called polymodifications, are evolutionarily conserved. Glycylation occurs in cells with cilia,while glutamylation occurs on a wider array of microtubule types including those formingcentrioles and the mitotic spindle. The ciliate Tetrahymena thermophila was used as anexperimental model to pursue the function of polymodifications. Previous studies showed thatmutations at sites of polymodifications on -tubulin results in Tetrahymena cells with paralyzed,and incompletely assembled cilia, that are arrested in cytokinesis probably due to incompletesevering of cortical longitudinal microtubule bundles. Thus, polymodification sites are requiredfor proper assembly and disassembly of diverse types of microtubules. Functional dissection ofthe respective roles of tubulin glutamylation and glycylation required identification of theresponsible enzymes. In the first part of the project we attempted to use biochemical means topurify the tubulin polyglycylase enzyme from cilia. It was possible to reconstitute the tubulinglycylase activity. A chromatographic procedure was developed which led to a 7000 foldpurification of the glycylase enzyme activity. Further identification of the catalytic subunit oftubulin glycylase was not possible due to low yield and stability. In the second part of theproject, we have discovered that evolutionarily conserved proteins with a domain homologous tothe catalytic center of tubulin tyrosine ligase (TTL) have tubulin polyglutamylase activity in vivoand in vitro. The Tetrahymena genome has 50 TTL domain loci, an extraordinarily large numberfor a unicellular organism. Among the predicted TTL-like (TTLL) proteins of Tetrahymena, weidentified two types of tubulin polyglutamylases, which differ in substrate preference (- vs. -tubulin). Even most closely related TTLL proteins of Tetrahymena, have distinct intracellularlocalizations. We showed that a portion of the less-conserved non-catalytic domain of onepolyglutamylase, Ttll6p, is required for its preferential targeting to cilia. Polyglutamylation of -tubulin by Ttll6p polyglutamylase stabilized cytoplasmic microtubules and strongly inhibitedciliary motility. On the other side, knockouts of TTLL1 and TTLL9 -tubulin glutamylase genesled to insufficient assembly of transverse and longitudinal cortical microtubules and inhibitedmaturation of newly assembled basal bodies. These studies reveal a novel mechanism forregulation of the microtubular cytoskeleton, based on spatially restricted tubulin-modifyingenzymes.