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.
The claret (ca) locus in Drosophila encodes a kinesin-related motor molecule that is required for proper distribution of chromosomes in meiosis in females and in the early mitotic divisions of the ...embryo. Here we demonstrate that a mutant allele of claret non-disjunctional (ca(nd)), non-claret disjunctional Dominant (ncadD), causes abnormalities in meiotic chromosome segregation, but is near wild-type with respect to early mitotic chromosome segregation. DNA sequence analysis of this mutant allele reveals two missense mutations compared with the predicted wild-type protein. One mutation lies in a proposed microtubule binding region of the motor domain and affects an amino acid residue that is conserved in all kinesin-related proteins reported to date. This region of the motor domain can be used to distinguish meiotic and mitotic motor function, defining an amino acid sequence criterion for classifying motors according to function. ncdD's mutant meiotic effect, but near wild-type mitotic effect, suggests that interactions of the ca motor protein with spindle microtubules differ in meiosis and mitosis.
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.
Motor proteins 1: kinesins Bloom, G S; Endow, S A
Protein profile,
1995, Letnik:
2, Številka:
10
Journal Article
Recenzirano
Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever ...increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles 17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292 or books 49, 227, 293 that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.
Recently, proteins have been identified that are required for proper distribution of chromosomes in meiosis and mitosis. Unexpectedly, several of these are microtubule motor proteins. This finding ...has prompted further investigation into the basis of meiotic and mitotic chromosome movement. The claret protein, or ncd, is one of the new motor proteins that may perform several functions in meiosis and early mitosis in Drosophila
Recent new information regarding the proteins required for proper distribution of chromosomes in meiosis has come from studies of Drosophila mutants. These studies reveal that proteins related to the ...microtubule motor protein, kinesin, function in meiotic chromosome segregation in Drosophila females. The two proteins identified thus far are likely to play very different roles in the process. The ncd protein is a spindle motor in meiosis but may perform a different role in the early mitotic divisions of the embryo. nod functions earlier in meiosis than ncd, prior to the meiotic divisions, and may be either chromosome or spindle associated. The identification of nod as a kinesin protein raises new questions regarding the distributive model of meiotic chromosome segregation.
The fungus gnat Sciara coprophila is a dipteran (two-winged) insect like the fruit fly Drosophila. But unlike Drosophila, Sciara undergoes programmed chromosome loss as part of its normal mechanism ...of sex determination during early development. Although the unusual behaviour of Sciara chromosomes during embryogenesis has received little attention over the past 20 years, the Saint Phalle and Sullivan have now elegantly extended previous observations in a study published in Development. Using laser-scanning confocal microscopy and fluorescence in situ hybridization, they have looked at chromosome elimination during early Sciara development. And their results can be understood in the context of current ideas about chromosome segregation in more "conventional" organisms.
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