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Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly.

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.