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 P-i 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 P-i 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 P-i 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 P-i. This result indicates that GTP hydrolysis and P-i 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 P-i in their E-sites. In addition, we have measured the E-site content of GTP and P-i 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-P-i cap model does not adequately explain yeast microtubule dynamic behavior.