Association of microtubules and neurofilaments in vitro is not mediated by ATP.

Abstract

Runge et al. [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435] found that mixtures of microtubules and neurofilaments formed a viscous, sedimentable complex when incubated at 37 degrees C for 20 min in the presence of ATP. They did not observe the high viscosities associated with the complex when the incubation was carried out in the absence of ATP. This paper reports an investigation of the roles of time and ATP in the formation of the complex. Microtubules assembled in a mixture containing GTP and neurofilaments prepared from bovine brain remained assembled for a shorter period of time than they did in similar solutions containing no neurofilaments. Adding ATP to the neurofilament-containing solutions, or doubling their GTP concentration, extended the time during which the microtubules remained assembled. These mixtures then became highly viscous. These phenomena resulted from the action of at least two enzymes present in the neurofilament preparation. A GTPase raised the GDP/GTP ratio, in the mixtures in which ATP was absent, to levels sufficient to cause disassembly of the microtubules. When ATP was present, a nucleotide diphosphokinase catalyzed regeneration of GTP from GDP while converting ATP to ADP. This process kept the GDP/GTP ratio low and delayed the disassembly of the microtubules. These results show that the apparent ATP dependence of formation of the microtubule-neurofilament complex observed by Runge et al. is attributable to a GDP-induced disassembly of microtubules rather than to a disruption of microtubule-neurofilament contacts. Those contacts can form in the absence of ATP.