DETECTION OF GTP AND P-I IN WILD-TYPE AND MUTATED YEAST MICROTUBULES - IMPLICATIONS FOR THE ROLE OF THE GTP GDP-P-I CAP IN MICROTUBULE DYNAMICS/

Microtubule dynamics are believed to be controlled by a stabilizing ca p 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-subu nit. However, it has been difficult to obtain convincing evidence to s upport this hypothesis because the quantity of GTP and P-i in the E-si te of assembled brain tubulin (the tubulin used in most studies thus f ar) is extremely low. In this study, we have measured the amount of GT P and P-i in the E-site of wild-type and mutated yeast assembled tubul ins. In contrast to brain microtubules, 6% of the tubulin in a wild-ty pe yeast microtubule contains a combination of E-site GTP and P-i. Thi s result indicates that GTP hydrolysis and P-i release are not coupled to dimer addition to the end of the microtubule and supports the hypo thesis 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 thou ght to interact with the nucleotide bound in the E-site. Previous stud ies have shown that microtubules containing these mutated tubulins hav e modified dynamic behavior in vitro. The results from these experimen ts indicate that the GTP or GDP-P-i cap model does not adequately expl ain yeast microtubule dynamic behavior.