Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism

The relationship between GTP hydrolysis and microtubule assembly has been investigated by using a rapid filtration method. Microtubules assembled from phosphocellulose-purified tubulin, double-labeled with [gamma-32P]- and [3H]GTP, were trapped and washed free of unbound nucleotide on glass fiber filters. The transient accumulation of microtubule-bound GTP predicted by uncoupled GTP hydrolysis models [Carlier & Pantaloni (1981) Biochemistry 20, 1918-1924; Carlier et al. (1987) Biochemistry 26, 4428-4437] during the rapid assembly of microtubules was not detectable under our experimental conditions. By calculating hypothetical time courses for the transient accumulation of microtubule-bound GTP, we demonstrate that microtubule-bound GTP would have been detectable even if the first-order rate constant for GTP hydrolysis were 4-5 times greater than the pseudo-first-order rate constant for tubulin subunit addition to microtubules. In a similar manner, we demonstrate that if GTP hydrolysis were uncoupled from microtubule assembly but were limited to the interface between GTP subunits and GDP subunits (uncoupled vectorial hydrolysis), then microtubule-bound GTP would have been detectable if GTP hydrolysis became uncoupled from microtubule assembly at less than 50 microM free tubulin, 5 times the steady-state tubulin concentration of our experimental conditions. In addition, during rapid microtubule assembly, we have not detected any microtubule-bound Pi, which has been proposed to form a stabilizing cap at the ends of microtubules [Carlier et al. (1988) Biochemistry 27, 3555-3559]. Also, several conditions that could be expected to increase the degree of potential uncoupling between GTP hydrolysis and microtubule assembly were examined, and no evidence of uncoupling was found. Our results are consistent with models that propose cooperative mechanisms that limit GTP hydrolysis to the terminal ring of tubulin subunits [e.g., O'Brien et al. (1987) Biochemistry 26, 4148-4156]. The results are also consistent with the hypothesis that a slow conformational change in tubulin subunits after GTP hydrolysis and Pi release occurs that results in destabilized microtubule ends when such subunits become exposed at the ends.