Reconstitution and Characterization of Budding Yeast γ-Tubulin Complex

Abstract

Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each cell division. γ-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast γ-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and theKdfor binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to γ-tubulin ring complexes found in higher eukaryotic cells.