Role of GTP in the assembly of microtubules.

Abstract

An in vitro system for the assembly of microtubules devoid of high-molecular-weight components, and also lacking the activities of ATPase and nucleoside diphosphate kinase has been developed. Using this system, it was demonstrated that a stoichiometric amount of GTP was sufficient to promote microtubule assembly and that the amount of inorganic phosphate liberated from GTP was equal to that of tubulin incorporated into the microtubules. ATP could only partially replace GTP at much higher concentrations, whereas GDP was completely ineffective when added alone and was inhibitory in the presence of GTP. GTP bound at the exchangeable site of tubulin was hydrolyzed to inorganic phosphate and GDP which became no longer exchangeable after polymerization of microtubules. On the other hand, GTP bound at the nonexchangeable site was not hydrolyzed during microtubule assembly. As reported previously (Arai, T. & Kaziro, Y. (1976) Biochem. Biophys. Research Commun. 69, 369–376), an unhydrolyzable analogue of GTP, guanyl-5′-yl imidodiphosphate (GMP-P(NH)P) could replace GTP in supporting the polymerization of microtubules. This suggested that the hydrolysis of GTP at the exchangeable site of tubulin is not a prerequisite of microtubule assembly. Using [3H]GMP-P(NH)P, it was shown that the incorporation of 3H-radioactivity into microtubules was equimolar to the amount of tubulin polymerized. The radioactivity incorporated into microtubules was recovered as [3H]GMP-P(NH)P. The micro tubules formed in the presence of GMP-P(NH)P were indistinguishable from those formed in the presence of GTP under electron microscopy but they had a markedly decreased sensitivity toward calcium ions. Also, a marked difference was observed between GTP- and GMP P(NH)P-promoted polymerization reactions in that the latter reaction was inhibited at much lower concentrations of GDP or calcium ions than the former. These observations suggest that the role of GTP in microtubule assembly is to give tubulin a conformation favorable for polymerization, and then to shift the equilibrium irreversibly toward polymerization through the hydrolysis of tubulin-bound GTP. The hydrolysis of GTP appears to be necessary for subsequent disassembly of microtubules, since GTP-assembled microtubules but not GMP-P(NH)P-assembled microtubules are sensitive to calcium-induced depolymerization.