Monomeric gamma -tubulin nucleates microtubules.

gamma-Tubulin is required for nucleation and polarized organization of microtubules in vivo. The mechanism of microtubule nucleation by gamma-tubulin and the role of associated proteins is not understood. Here we show that in vitro translated monomeric gamma-tubulin nucleates microtubules by lowering the size of the nucleus from seven to three tubulin subunits. In capping the minus end with high affinity (10(10) m(-1)) and a binding stoichiometry of one molecule of gamma-tubulin/microtubule, gamma-tubulin establishes the critical concentration of the plus end in the medium and prevents minus end growth. gamma-Tubulin interacts strongly with beta-tubulin. A structural model accounts for these results.

Cofactor A is a molecular chaperone required for beta-tubulin folding: functional and structural characterization.

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the chaperonin containing TCP-1 (CCT) and, in the case of alpha- and beta-tubulin additional protein cofactors. We recently identified one of these cofactors, termed cofactor A, that is required for the proper folding of the beta-tubulin chain [Gao et al. (1994) J. Cell. Biol. 125, 989-996]. We show here that cofactor A, a monomeric protein that has no measurable affinity for nucleotides, is a highly conserved protein among vertebrates. Its NH2-terminal region is essential for the structural integrity of the protein and consequently for its activity. We demonstrate that cofactor A does not interact with CCT nor does it affect the intrinsic ATPase activity of CCT, alone or in the presence of different target proteins. Thus, unlike GroES, cofactor A does not modulate or coordinate ATP hydrolysis. It does not act as a nucleotide exchange factor or a catalyst in tubulin folding. Rather, we demonstrate that cofactor A participates in the tubulin folding process by interacting with a folding intermediate of beta-tubulin that is released from CCT. Our data imply that cofactor A is a chaperone involved in tubulin folding.