In vitro microtubule assembly regulation by divalent cations and nucleotides

Mg2+ and guanosine 5'-triphosphate (GTP) are usually used to assemble microtubule protein (tubulin + microtubule-associated proteins) or tubulin into microtubules in vitro. Recently, it has been shown that Mn2+ will substitute for Mg2+ in inducing pure tubulin + GTP to assemble into microtubules. We find that Mn2+ also substitute for Mg2+ in inducing two-cycle calf microtubule protein (MTP) + GTP to assemble into microtubules. Zn2+ or Co2+ induces MTP + GTP to form sheets with more than 13 protofilaments. We find that Co2+ also substitutes for Zn2+ in inducing tubulin to form 200-nm tubules similar to those reported with Zn2+. To learn whether metal-induced assembly is due to direct binding and/or a metal-GTP complex, metal-induced assembly of MTP and tubulin was studied in the presence of CrIIIGTP or taxol, two probes which promote assembly in the absence of added GTP. With CrGTP, Mg2+ and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) were required for optimal assembly into microtubules, and Mn2+ could substitute for Mg2+. MTP incubated with Zn2+ and CrGTP assembled into sheets, shorter than but similar to those induced by Zn2+ + GTP. Mg2+-induced microtubules and Zn2+-induced sheets contained 0.45 mol of [8-3H]GTP/mol of tubulin if assembled from [8-3H]GTP and a mixture of 0.25 mol of [8-3H]CrGTP, 0.1 mol of [8-3H]GDP, and 0.05 mol of [8-3H]CrGDP/mol of tubulin if assembled from [8-3H]CrGTP. Zn2+ induced taxol-treated MTP to form sheets. Sheets were also induced from tubulin + Zn2+ and either CrGTP or taxol. These studies suggest that the Zn2+-induced structures are not due to a Zn-GTP complex and that Mg2+ does not promote assembly only through a Mg-GTP complex.