Drosophila Ncd Research Articles

Modeling Study of the Dynamics of Kinesin-14 Molecular Motors.

Kinesin-14s constitute a subfamily of the large superfamily of adenosine triphosphate-dependent microtubule-based motor proteins. Kinesin-14s have the motor domain at the C-terminal end of the peptide, playing key roles during spindle assembly and maintenance. Some of them are nonprocessive motors, whereas others can move processively on microtubules. Here, we take budding yeast Cik1-Kar3 and human HSET as examples to study theoretically the dynamics of the processive kinesin-14 motor moving on the single microtubule under load, the dynamics of the motor coupled with an Ndc80 protein moving on the single microtubule, the dynamics of the motor moving in microtubule arrays, and so on. The dynamics of the nonprocessive Drosophila Ncd motor is also discussed. The studies explain well the available experimental data and, moreover, provide predicted results. We show that the processive kinesin-14 motors can move efficiently in microtubule arrays toward the minus ends, and after reaching the minus ends, they can stay there stably, thus performing the function of organizing the microtubules in the bipolar spindle into polar arrays at the spindle poles.

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s

Drosophila melanogaster kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested here is that Kar3Vik1 and Kar3Cik1, as well as Ncd, use a common ATPase mechanism for force generation even though the microtubule interactions for both Ncd heads are modulated by nucleotide state. The presteady-state kinetics and computational modeling establish an ATPase mechanism for a powerstroke model of Ncd that is very similar to those determined for Kar3Vik1 and Kar3Cik1, although these heterodimers have one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain whose interactions with microtubules are not modulated by nucleotide state but by strain. The results indicate that both Ncd motor heads bind the microtubule lattice; two ATP binding and hydrolysis events are required for each powerstroke; and a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. Note that unlike conventional myosin-II or other processive molecular motors, Ncd requires two ATP turnovers rather than one for a single powerstroke-driven displacement or step. These results are significant because all metazoan kinesin-14s are homodimers, and the results presented show that despite their structural and functional differences, the heterodimeric and homodimeric kinesin-14s share a common evolutionary structural and mechanochemical mechanism for force generation.

Novel Kinesin Regulators of Gamma-TuRC

Mitotic spindle assembly is a critical control point in eukaryotic chromosome segregation. In fission yeast conserved mitotic kinesin-like proteins (Klps) Kinesin-14 and Kinesin-5 are emerging as novel regulators of the γ-tubulin ring complex (γ-TuRC) MTOC. While these Klps typically participate in microtubule roles for spindle assembly, alternatively in fission yeast they localize to γ-TuRC and when co-deleted a bipolar spindle forms. Our analysis by genetics, biochemistry, cell biology and nanotechnology provides new knowledge on mitotic mechanisms in S. pombe. Previous work from our lab and others uncovered a novel functional relationship between Kinesin-14 Pkl1 and all proteins of the γ-tubulin small complex (γ-TuSC). We demonstrated that Pkl1 binds γ-TuRC through Motor and Tail interactions to directly regulate its function. A Pkl1 Tail peptide is sufficient in vitro and disrupts γ-TuRC structure by removal of γ-tubulin subunits (Cell Cycle 2013), an action that is reversible. New data from our lab indicates that the essential Kinesin-5 Cut7 is actually dispensable in the absence of Pkl1 with which it shares an important antagonistic MTOC regulatory relationship. Cut-7 associates with FPLC purified γ-TuRC and binds to γ-tubulin similarly as Pkl1. We demonstrate in vivo that Pkl1 blocks microtubule nucleation from γ-TuRC and additionally influences spindle microtubule numbers and microtubule overlap. Kinetochore-based microtubule motors Klp5 and Klp6 may facilitate spindle organization, since triple mutants of Pkl1, Cut7 and either Kinesin-6 Klp9 or Kinesin-14 Klp2 are viable. Previously we showed in vivo that human Kinesin-14 HSET, but not Drosophila Ncd, functionally replaces Pkl1 and more recently that human γ-TuSC proteins GCP2 and GCP3 replace fission yeast Alp4 and Alp6 (JCS 2013). Our research highlights conserved in vivo functions of γ-TuSC and provides a new model of spindle assembly in S. pombe.

Kinesin-14: A League of their Own

Kinesin-14 represents a subfamily of kinesins that are nonprocessive, promote microtubule (MT) minus-end-directed force generation, and contain C-terminal motor domains that are dimerized through an N-terminal coiled-coil. Unlike the well-known N-terminal motor domain kinesins that use an asymmetric hand-over-hand mechanism for MT plus-end-directed processive stepping, Kinesin-14s use a MT minus-end-directed powerstroke to generate force to crosslink and slide one MT relative to another. While most Kinesin-14s are homodimers like Drosophila Ncd, S. cerevisiae Kar3Vik1 is a heterodimer. The C-terminal domain of Vik1 exhibits the structural fold of a kinesin motor domain, binds MTs independent of Kar3, yet lacks a nucleotide-binding site. Furthermore, Kar3Vik1 binds across adjacent MT protofilaments, a non-canonical MT binding configuration. The results indicate that Kar3Vik1 collides with the MT through Vik1, promoting MT binding by Kar3. The tight binding of Kar3 destabilizes the Vik1 interaction with the MT, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with the rotation of the coiled-coil stalk, and post-powerstroke ATP hydrolysis is independent of Vik1 providing additional evidence that Vik1 rotates with the coiled-coil during the powerstroke. Detachment of Kar3Vik1 from the MT completes the cycle and allows the motor to return to its initial conformation. Supported by NIH GM54141.

Metamorphic Pattern Formation and Deformation: In Vivo and In Vitro Mechanisms

Mitotic spindle formation into a bipolar structure suitable for chromosome segregation requires reorganization of the interphase microtubule cytoskeleton. A multi-protein complex at spindle poles acts as a microtubule organizing center (MTOC). Microtubules are assembled from this site through the addition of α/β-tubulin heterodimers onto a template complex containing γ-tubulin (γ-TuSC) imbedded into a larger macromolecular ring (γ-TuRC). Our goal is to apply insights on patterning biological polymers in vivo to development of hybrid biosynthetic systems capable of utilizing microtubules in self-assembling metamorphic patterns including parquet deformation behavior. Dynamic patterning has applications in biosensing, materials design and new nanomanufacturing paradigms. Building off of recent structural insights into γ-TuSC and GCP4 with our own detailed genetic analysis, site-directed mutagenesis, cross-species functional studies and biochemical purification and nucleation assays we provide novel insights on MTOC structural requirements to nucleation. Additionally we have identified an associated regulatory mechanism utilized by a subset of Kinesin-14 members for targeting and regulatory interference at poles (TRIP) distinct from microtubule targeting elements found in other Kinesin-14 members such as Drosophila Ncd. Our in vivo analysis supports application of the recent Kollman-Agard structure as a general eukaryotic model however with species-specific protein and domain constraints as well as contact sites for Kinesin-14 regulation of γ-TuRC. Our findings have broad application towards a general understanding of cellular MTOC machinery and reiterates the flexibility of Klps to localize to multiple spindle compartments and participate in a diversity of cellular roles. Natural cellular machines provide tools and inspiration for biomimicry and for resolution of societal grand challenges through redirected applications. Using tools available through the semiconductor industry and materials science we are generating artificial centrosomes (ACENs) from nanoparticles to act as simplified nucleation centers for use in synthetic self-assembly paradigms.

Klp Navigation of Spindle Assembly from Poles

Spindle assembly is the crucial initiating step in the mitotic mechanism. Its formation utilizes coordinated actions of Kinesin-like proteins (Klps) at poles, on microtubules and at chromosomes. We previously identified a novel conserved Kinesin-14 pole-based mechanism for spindle assembly by Klp binding to the γ-tubulin small complex (γ-TuSC) microtubule organizing center, (MTOC). Changes to the γ-TuSC MTOC in fission yeast alter microtubule dynamics to regulate bipolarity and both human and fission yeast Kinesin-14 Klps can regulate this mechanism. The Kinesin-14 Pkl1 motor domain associates with residues in helix 11 of γ-tubulin at a novel Klp binding site. The Kinesin-14 Pkl1 Tail domain is distinct from that of the well-characterized Drosophila Ncd, replacing microtubule association sites with specialized elements for spindle pole targeting and regulation of the γ-TuSC. Thus unlike Drosophila Ncd that crosslinks microtubules to stabilize mitotic spindle assembly, Kinesin-14 Pkl1 regulates assembly through the γ-TuSC. Klps can be classified into 14 families. Members of two ubiquitous families, Kinesin-14 and opposing Kinesin-5 Klps, each exhibit distinctions at multiple levels amongst members, including domain elements, functional mechanisms and spindle localization. In fission yeast, Kinesin-5 also localizes to spindle poles, consistent with the hypothesis that similar localization is needed to optimally counterbalance forces by these two Klp families. Although the stoichiometry of proteins within γ-TuSC is known and conserved in eukaryotes little is understood of the γ-TuSC mechanism. Cross-species analysis is defining shared and distinct functions and novel insights into the γ-TuSC interactome and the role of associating Klps in regulating its mechanism for spindle assembly. Our studies apply site directed mutagenesis, genetics, yeast two hybrid, biochemistry, timelapse microscopy and bioinformatic/structural approaches with novel reagents we have developed for Klp and γ-TuSC analysis.

Cross-Species Analysis of Kinesin-14s: Human HSET Functions in Fission Yeast to Regulate Spindle Bipolarity

The discovery of Kinesin-like proteins in 1990, five years after conventional Kinesin, has blossomed into the identification of hundreds of Klps that are classified into fourteen families and one orphan family. Not all Klp families are conserved between eukaryotes, however the Kinesin-14 Klps are ubiquitous and key regulators of bipolar spindle assembly and microtubule dynamics and organization. Kinesin-14 Klps associate with microtubules through ATP-dependent binding of their motor domains, but an additional ATP-independent microtubule binding site in the stalk domain is proposed to allow bundling of microtubules. Kinesin-14 members represent some of the most extensively studied Klps, however limited analysis has been done to examine conserved domains or mechanisms. Kinesin-14s share a domain structure in the stalk that consists of an N-terminal region followed by a region of coiled coil. They can vary drastically in size and share minimal sequence similarities. Features of a second microtubule-binding domain are unknown as well as regulatory elements. Most recently this lab has determined that Kinesin-14/γ-tubulin interactions at the γ-TuSC MTOC can regulate bipolarity. Other genetic and biochemical analysis indicates that Kinesin-14 interactions with other MTOC proteins also occur. We are using deletion analysis, site-directed mutagenesis and chimeric Kinesin-14 proteins with human HSET and Drosophila Ncd to relate structural and sequence domains in Kinesin-14s to their functional importance. Twenty-five Kinesin-14 constructs have been generated and are helping to define the minimal Kinesin-14 and to separate domains for bipolar spindle assembly versus later mitotic roles. Length requirements of the coiled-coil domain are also being examined as a critical component in models that emphasize microtubule cross-linking by Kinesin-14s. This work provides insights into Kinesin-14 functional needs and unique tools for assessing Kinesin-14 interactions with tubulin and non-tubulin proteins in conserved molecular mechanisms.

Coiled Coil in the Stalk Region of ncd Motor Protein Is Nonlocally Sustained

The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192-346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The alpha helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high alpha helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252-272), (276-330), and (336-346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332-369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for alpha-helix continuity. This would make the region (336-346), as the head-stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility.

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.

Cik1 Targets the Minus-End Kinesin Depolymerase Kar3 to Microtubule Plus Ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

Archived gels as a tool for identification of protein complexes: Polo kinase cofractionates with Drosophila 205-kDa MAP and ncd in mitotic embryonic extracts

Archived gels as a tool for identification of protein complexes: Polo kinase cofractionates with Drosophila 205-kDa MAP and ncd in mitotic embryonic extracts

The ATPase Cross-bridge Cycle of the Kar3 Motor Domain: IMPLICATIONS FOR SINGLE HEAD MOTILITY

Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with Ncd showed that both motor domains were required for ATP-dependent motor domain detachment from the microtubule. We have initiated a series of experiments to compare the mechanistic requirements for Kar3 motility in direct comparison to Ncd. The results presented here show that the single motor domain of Kar3 (Met(383)-Lys(729)) exhibits characteristics similar to monomeric Ncd. The microtubule-activated steady-state ATPase cycle of Kar3 (k(cat) = 0.5 s(-1)) is limited by ADP release (0.4 s(-1)). Like monomeric Ncd, Kar3 does not readily detach from the microtubule with the addition of MgATP. These results show that the single motor domain of Kar3 is not sufficient for ATP-dependent microtubule dissociation, suggesting that structural elements outside of the catalytic core are required for the cyclic interactions with the microtubule for force generation.

A novel C-terminal kinesin subfamily may be involved in chromosomal movement in Caenorhabditis elegans

C-terminal kinesin motor proteins, such as the Drosophila NCD and yeast KAR3, are involved in chromosomal segregation. Previously we have described two orthologs of NCD in Caenorhabditis elegans, KLP-3 and KLP-17, which also participate in chromosome movement. Here we report cDNA cloning of klp-15 and klp-16, and the expression pattern of the genes encoding C-terminal motor kinesins including klp-15 and klp-16. Interestingly KLP-15 and KLP-16 form a unique class of C-terminal kinesins, distinct from the previously known C-terminal motors in other organisms. Using in situ hybridization and RNA interference assay, we show that although all of these motors mediate chromosome segregation, they do so in a combination of unique and overlapping manners, suggesting a complex hierarchy of kinesin motor function in metazoans.

CDNA Cloning and Expression of a C-Terminus Motor Kinesin-like Protein KLP-17, Involved in Chromosomal Movement in Caenorhabditis elegans

Members of the kinesin protein family transport intracellular cargo to their correct cellular destination. Previously we have characterized the klp-3 gene from Caenorhabditis elegans, which encodes an ortholog of the retrograde C-terminus kinesin motors, such as Drosophila NCD, and yeast KAR3, involved in the chromosomal movement. Here we report the cloning of a full-length klp-17 cDNA in C. elegans, encoding a C-terminus kinesin of 605 amino residues. KLP-17 sequence defines a novel phylogenetic group, distinct from the NCD/KAR3 family. Interestingly, the klp-17 gene transcript is restricted to the nuclear compartment, as deduced by the RNA in situ hybridization in embryos. The klp-17::gfp-expressing transgenic animals do not display any GFP fluorescence signal, but expression of the extra chromosomal arrays cause production of abnormal males, and embryos with morphological defects and lethality in the progeny. Similarly, the klp-17 RNA interference assay results in embryonic death, arrested embryos, and polyploid cells. Thus, KLP-17 represents a new motor protein that mediates chromosome movement, essential for cell divisions during metazoan development.

Solution structures of dimeric kinesin and ncd motors.

The dimeric structure of the members of the kinesin family of motor proteins determines the individual characteristics of their microtubule-based motility. Crystal structures for ncd and kinesin dimers, which move in opposite directions on microtubules, show possible states of these dimers with ADP bound but give no information about these dimers in solution. Here, low-angle X-ray and neutron scattering were used to investigate their solution structures. Scattering profiles of Drosophila ncd 281-700 (NCD281) and human kinesin 1-420 (hKIN420) were compared with models made from the crystallographically determined structures of NCD281 and rat kinesin 1-379 (rKIN379). From the low-angle region it was found that the radius of gyration (Rg) of NCD281 is 3.60 +/- 0.075 nm, which is in agreement with the crystallography-based model. Scattering by longer ncd constructs (NCD250 and NCD224) is also well fit by the appropriate crystallography-based models. However, the measured Rg of hKIN420, 4.05 +/- 0.075 nm, is significantly smaller than that of the crystallography-based model. In addition, the overall scattering pattern of NCD281 is well fit by the model, but that of hKIN420 is poorly fit. Model calculations indicate that the orientation of the catalytic cores is different from that observed in the rKIN379 crystal structure. Like the crystal structure, the best-fitting models do not show 2-fold symmetry about the neck axis; however, their overall shape more resembles a mushroom than the "T"-like orientation of the catalytic cores found in the crystal structure. The center of mass separations of the catalytic cores in the best-fitting models are 0.7-1 nm smaller than in the crystal structure.

Crystal structure of the motor domain of the kinesin-related motor ncd.

Microtubule-based ATPases of the kinesin superfamily provide the motile force for many animated features of living cells. Kinesin motors differ in their direction of movement along microtubules. Kinesin and ncd, a kinesin-related motor involved in formation and maintenance of mitotic and meiotic spindles, move in opposite directions along microtubules, even though their motor domains are 40% identical in amino-acid sequence. Here we report the crystal structure of the MgADP complex of the Drosophila ncd motor domain determined to 2.5A by X-ray crystallography, and compare it to the kinesin structure. The ncd and kinesin motor domains are remarkably similar in structure, and the locations of conserved surface amino acids suggest these motors share a common microtubule-binding site. Moreover, structural and functional comparisons of ncd, kinesin, myosin and G proteins indicate that these NTPases may have a similar strategy of changing conformation between NTP and NDP states. We propose a general model for converting a common gamma-phosphate-sensing mechanism into opposite polarities of movement for kinesin and ncd.

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.

Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.

The kinesin heavy chain and the ncd (non-claret disjunctional) gene product of Drosophila are microtubule-associated motor proteins related by sequence similarity within an approximately 340-aa domain. Despite the sequence similarity, the kinesin heavy chain and ncd protein move in opposite directions on microtubules. To investigate the molecular basis for direction of movement, we created a series of truncated kinesin heavy chain and ncd proteins. We found that the conserved domain of both proteins has microtubule motor activity, although the efficiency with which ATP hydrolysis is coupled to microtubule movement declines dramatically with increasing truncation. Further, the direction of movement is intrinsic to the conserved motor domains, rather than being a consequence of domain organization or adjacent sequences.