MECHANISM OF SEGREGATION OF MODIFIED TUBULIN IN VIVO

Microtubules (MTs) are cytoskeletal elements found in all eukaryotic cells, where they function in a variety of motile and morphogenetic events. All MTs are polymers of tubulin protomers, which are long-lived cellular proteins whose sequence is remarkably constant among different organisms and cell types. The long-range goal of the research proposed is to understand what biochemical alterations in tubulin or its associated proteins can bring about changes in the function of MTs. In particular, post-transcriptional modifications would seem to be good candidates for alterations that could rapidly and reversibly affect MT organization or function. Tubulin is subject to a unique pair of post-translational modifications, called tyrosination/detyrosination, in which a tyrosine residue is added to or removed from the C-terminus of the alpha chain. We previously found that MTs enriched in tyrosinated or detyrosinated tubulin composed complementary subsets of cellular MTs, and that these tubulin species become segregated by a cyclic mechanism involving tyrosination/detyrosination and tubulin polymerization/depolymerization. We have also demonstrated that MTs enriched in detyrosinated tubulin exhibit unusual properties and dynamics. Here we propose further studies of this unusual population of stable, detyrosinated MTs, and a determination of which properties of these MTs are a function of the presence of detyrosinated protomers. In addition, we will perform detailed studies on the enzymes that tyrosinate (tubulin tyrosine ligase) and detyrosinate (tubulin carboxypeptidase) alpha- tubulin; purifying each, assaying its activity and levels during MT- mediated cellular events, deriving cDNA clones to each and comparing its properties to those of other related enzymes. Finally, characterization of the enzymes and the unusual MTs they create will allow us to develop chemical and anti-sense RNA inhibitors of each enzyme, in order to determine the role of the tyrosination/detyrosination cycle in MT function. Our proposed work on this unique cycle of post-translational modification will enable us to better understand the function of MTs during differentiation and cell division in both normal and pathological settings.