ANALYSIS OF TUB4P, A YEAST GAMMA-TUBULIN-LIKE PROTEIN - IMPLICATIONS FOR MICROTUBULE-ORGANIZING CENTER FUNCTION

gamma-Tubulin is a conserved component of microtubule-organizing cente rs and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin tha t is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is locali zed to the spindle pole body (Sobel, S.G., and M. Snyder. 1995. J. Cel l Biol. 131:1775-1788). We have characterized the expression of TUB$, the association of Tub4p with the spindle pole body, and its role in m icrotubule organization. Tub4p is a minor protein in the cell, and exp ression of TUB4 is regulated in a cell cycle-dependent manner. Wild-ty pe Tub4p is localized to the spindle pole body, and a Tub4p-green fluo rescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplas mic and spindle pole body forms of Tub4p. Perturbation of Tub4p functi on, either by conditional mutation or by depletion of the protein, res ults in spindle as well as spindle pole body defects, but does not eli minate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cell s duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer i nsight into the role of gamma-tubulin in microtubule-organizing center function.