An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays.
The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis. The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends. The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain. In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region. Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg(2+)-ATPase activity and binds microtubules in pelleting assays. In contrast to near full-length ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays. Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis. The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle. If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.
Chandra
R
R
Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
Endow
S A
SA
Salmon
E D
ED
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D004058:Diffusion; D004330:Drosophila; D029721:Drosophila Proteins; D005898:Glass; D066298:In Vitro Techniques; D016547:Kinesins; D008868:Microtubule Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology
D029721:Drosophila Proteins; D008868:Microtubule Proteins; C066696:ncd protein, Drosophila; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D016547:Kinesins