2012 ASCB Annual Meeting abstracts

γ-Tubulin nucleates microtubules (MTs) in all eukaryotes and acts in complex with an array of related accessory proteins. It exists in at least two types of complexes inside cells: a ~300kDa γ-Tubulin Small Complex (γTuSC), which can assemble to form a ~2.2MDa γ-Tubulin Ring Complex (γTuRC). In vitro MT nucleation assays have shown that the γTuRC is a more potent nucleator than γTuSC. Interestingly, pure human γ-tubulin can form filaments in vitro that are also much more active than γTuSC. We have examined the structure-function relationship between the configuration of γ-tubulins in these complexes and their ability to nucleate MTs using high-resolution electron microscopic reconstructions of the complexes and in vitro MT nucleation assays. We find that complexes in which the γ-tubulins make lateral contacts like those made by αβ-tubulins in the MT wall are the best nucleators. Our finding that the γTuSC (Tub4 complex) of S. cerevisiae can assemble spontaneously into γTuRC-like rings with 13-fold symmetry, but without a precise MT-lattice-like arrangement of γ-tubulins, suggested a mode of regulation for γTuRC activity. The flexibility observed in one of the "arms" of the V-shaped γTuSC suggests that a conformational change is required to bring the γ-tubulins into an optimal position for achieving MT nucleation. We are exploring this possibility. We have also found that the S. cerevisiae γ-tubulin ring complexes show much greater activity with S. cerevisiae αβtubulin than with pig brain tubulin.