Detection of GTP and Pi in wild-type and mutated yeast microtubules: implications for the role of the GTP/GDP-Pi cap in microtubule dynamics.

Microtubule dynamics are believed to be controlled by a stabilizing cap of tubulin dimers at microtubule ends that contain either GTP or GDP and Pi in the exchangeable nucleotide site (E-site) of the beta-subunit. However, it has been difficult to obtain convincing evidence to support this hypothesis because the quantity of GTP and Pi in the E-site of assembled brain tubulin (the tubulin used in most studies thus far) is extremely low. In this study, we have measured the amount of GTP and Pi in the E-site of wild-type and mutated yeast assembled tubulins. In contrast to brain microtubules, 6% of the tubulin in a wild-type yeast microtubule contains a combination of E-site GTP and Pi. This result indicates that GTP hydrolysis and Pi release are not coupled to dimer addition to the end of the microtubule and supports the hypothesis that microtubules contain a cap of tubulin dimers with GTP or Pi in their E-sites. In addition, we have measured the E-site content of GTP and Pi in microtubules assembled from two yeast tubulins that had been mutated at residues T107 and T143 in beta-tubulin, sites thought to interact with the nucleotide bound in the E-site. Previous studies have shown that microtubules containing these mutated tubulins have modified dynamic behavior in vitro. The results from these experiments indicate that the GTP or GDP-Pi cap model does not adequately explain yeast microtubule dynamic behavior.