A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity

Abstract

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncd(D) retains mitotic, but not meiotic, function. The predicted Ncd(D) mutant protein contains a V556→F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and Ncd(D) proteins. A GST-Ncd(D) fusion protein translocated microtubules ~ 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an Ncd(D) motor domain protein was reduced ~ 3-fold and an ~ 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The K(m) for ATP and basal rate of ATP turnover were, in contrast, similar for the Ncd(D) mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant Ncd(D) motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of Ncd(D) translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the Ncd(D) motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.