NCD activation of tubulin polymerization.

Tubulin dimer (tT) was purified from turkey erythrocytes. The motor domain of Drosophila non-claret disjunctional protein, NCD(335-700), was expressed in E. coli and purified. At 37 degrees C in the presence of GTP, the rate of polymerization of tT to microtubule (tMt) is accelerated over threefold by the presence of NCD(335-700). At 10 degrees C, the rate of tT polymerization is increased from zero, within experimental error, in the absence of NCD(335-700) to rates near those observed at 37 degrees C when NCD(335-700) is present. The NCD(335-700) concentration dependence of the rate indicated the reactive species was NCD(335-700)(n).tT, with n approximately 2. At 10 degrees C in the absence of GTP, polymerization does not occur, but tT activates NCD(335-700) MgATPase activity 10-fold. For the same conditions, using mians-NCD(335-700), which is modified with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid, the apparent K(D) for binding to tT is 2.3 x 10(-5) M in the presence of MgADP. Replacing ADP with AMPPNP or ATP has a negligible effect on K(D). Mians-NCD(335-700) binding to tMt is 10-fold stronger than to tT. The above data indicate NCD(335-700) binds at a functional site on tT. The stoichiometry is consistent with the formation of NCD(335-700)(2).tT which in vitro accelerates self-assembly initiation and/or polymerization by binding a second tT in a position favorable for tubulin-tubulin interaction. The data suggest that in vivo functional NCD binding to microtubule involves one motor domain binding to alpha- and beta-subunits at the interface of two different tubulin dimers in a protofilament.