The relationship between cobalt binding to tubulin and the stimulation of assembly.

Abstract

Co(II), which induces the assembly of tubulin in the absence of associated proteins, binds to multiple sites on the protein in a low ionic strength medium.However, in the presence of concentrations of buffer and GTP used in assembly reactions, the number of sites occupied by Co(I1) is drastically reduced.At GTP and Co(I1) concentrations where the major polymerized products are broad sheets of protofilaments, about 3 Co(II) atoms are bound/tubulin dimer.Increasing the GTP concentration lowers the amount of assembly, increases the proportion of microtubules to sheets, and decreases the amount of Co(I1) bound.It is concluded that the binding of free Co(II) is necessary to stimdate assembly and the less Co(II) bound, the more likely is the formation of microtubules.Tubulin containing 1 tightly bound Co(I1) can be isolated by passing tubulin preincubated with an excess of Co(II) through phosphocellulose.Only 50% of the tight Co(I1) binding can be prevented by Mg(I1) or Mn(II), but 100% is prevented by Zn(II), Cd(II), and Cu(1).Moreover, Mg(1I) can only displace a fraction of the bound Co(I1).The Mg(I1) content of tubulin is reduced after the cobalt is bound.Tubulin containing 1 tightly bound cobalt can assemble into microtubules and sheets if either more cobalt or taxol is added.The Mg(I1) and Co(I1) contents of cold-soluble assembled products are approximately 1 atom/tubulin dimer, respectively, suggesting that the Co(II) is not situated at the Mg(I1) site and that only tubulin containing 1 Mg(II)/dimer assembles into protofilamentous structures.Tubulin is a metalloprotein, containing 1 atom of tightly bound Mg(II)/dimer (1-3).The role this cation plays in the structural integrity and/or the self-assembly properties of tubulin has not been elucidated.Although it is possible that the Mg(I1) is complexed by the nonexchangeable nucleotide, no experimental evidence has been presented to demonstrate this.In addition to this bound metal, additional Mg(I1) is required to achieve maximal self-assembly rates (2).Mn(I1) also supports the in vitro formation of microtubules (4,5) and can displace the bound Mg(I1) (4).Co(I1) and Zn(I1) bind tightly to tubulin (6) and promote self-assembly, but the