NCD activation of tubulin polymerization

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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°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°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°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≈2. At 10°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 2.3×10 −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 α- and β-subunits at the interface of two different tubulin dimers in a protofilament.