A POINT MUTATION IN THE MICROTUBULE-BINDING REGION OF THE NCD MOTOR PROTEIN REDUCES MOTOR VELOCITY

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oo cytes 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 th e putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and Ncd(D) pr oteins. A GST-Ncd(D) fusion protein translocated microtubules similar to 10-fold more slowly than the corresponding wild-type protein in gli ding assays. The maximum microtubule-stimulated ATPase activity of an Ncd(D) motor domain protein was reduced similar to 3-fold and an simil ar to 3-fold greater concentration of microtubules was required for ha lf-maximal stimulation of ATPase activity, compared with the correspon ding 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 mo tor domain proteins. Pelleting assays demonstrated that the binding of the mutant Ncd(D) motor protein to microtubules was reduced in the ab sence of nucleotide, relative to wild-type. The reduced velocity of Nc d(D) translocation on microtubules is therefore correlated with reduct ions in microtubule-stimulated ATPase activity and affinity of the mut ant motor for microtubules. The characteristics of the Ncd(D) motor ex plain its meiotic loss of function, and are consistent with partial mo tor activity of Ncd being sufficient for its mitotic, but not its meio tic, role.