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 oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD 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 NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD 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 NcdD 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.
Moore
J D
JD
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Song
H
H
Endow
S A
SA
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics; D016547:Kinesins; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011993:Recombinant Fusion Proteins; C066696:ncd protein, Drosophila; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D016547:Kinesins