Binding of ATP to tubulin.

Abstract

Microtubules are present in all eukaryotic cells and participate in a variety of cellular processes1. From recent studies it has become clear that nucleotides have a central role in the assembly of tubulin into microtubules2–5. The tubulin dimer binds two moles of guanine nucleotide: one (at the E-site) which can exchange with exogenous nucleotide and one (at the N-site) which does not exchange6. E-site GTP is hydrolysed during the assembly process4,7,8. Microtubule assembly can be induced by ATP through a contaminating nucleoside diphosphokinase activity that regenerates GTP following its hydrolysis—this is associated with tubulin polymerization4,9,10. Recent reports suggest that ATP has other effects on the assembly kinetics11–13, and we have shown14,15, using tubulin preparations lacking the nucleoside diphosphokinase activity, that ATP may play a critical part in the regulation of microtubule formation by binding to tubulin at a site which is distinct from the N- and E-sites. Kinetic data suggest a model in which ATP can act at the level of nucleation to stimulate assembly15. We now report the first direct evidence for the binding of ATP to tubulin and show that the corresponding dissociation constant is in a range of physiological significance.