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Query: UNIPROT:P06889 (Mol)
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We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.
Mol Biol Cell 1996 Oct
PMID:Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function. 889 67

A general mechanism for polymerase translocation is elaborated. The central feature of this mechanism is that a rapid translocational equilibrium is established after each cycle of nucleoside monophosphate incorporation such that the polymerase distributes itself by diffusional sliding between all accessible positions on the template with relative occupancy determined by relative free energy. While alternative models for translocation have not been fully developed, much of the language currently used to describe this step suggests an active mechanism coupled to conformational transitions in the polymerase. For example, a recent study of force generation by Escherichia coli RNA polymerase during transcription suggests that it is a mechanoenzyme analogous to kinesin of myosin motor proteins. While the proposed mechanism does not rule out conformational transitions during polymerase translocation, it suggests that they may be unnecessary and that translocation can be explained in terms of the affinity of the active site for nucleoside triphosphate and the relative free energies of the polymerase bound at different positions on the template. This mechanism makes specific predictions which are borne out experimentally with polymerases as distinct as E. coli DNAP I, phage T7 RNAP, and E. coli RNAP.
J Mol Biol 1997 Jan 10
PMID:A model for the mechanism of polymerase translocation. 899 20

Kinesin and ncd (non-claret disjunctional) are microtubule associated motor proteins which share several structural features: both motors are dimers; each monomer is composed of a stalk region, a cargo binding domain and a motor domain; the motor domains have approximately 41% sequence identity. Despite these similarities the two motors have strikingly different movement properties: kinesin is a plus-end directed molecular motor, while ncd is minus-end directed. Here we compare the structure and the microtubule-binding properties of these oppositely directed molecular motors. We determined the three-dimensional structure of tubulin sheets decorated with the motor domains of either kinesin or ncd to a resolution of < 20 A by negative stain electron microscopy and tilt series reconstruction. Comparisons with a control structure of tubulin alone revealed that in both cases the motor domain binds to the outer crest of a single protofilament making contacts with both alpha and beta tubulin. Despite their opposite directionality, the geometry of attachment of the motor domain to the protofilament in the presence of AMP-PNP is very similar for both motors. These data rule out models for directionality which have the motors binding in an opposite orientation to the microtubules. Binding of the ncd as well as the kinesin motor domain appears to induce conformational changes in tubulin. This observation suggests an active role of tubulin in motor movement and/or in the determination of directionality.
J Mol Biol 1997 Feb 07
PMID:Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. 904 48

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.
Mol Biol Cell 1997 Jun
PMID:Centromere position in budding yeast: evidence for anaphase A. 920 8

Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.
Mol Biol Cell 1997 Jun
PMID:Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors. 920 13

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.
Mol Biol Cell 1997 Jun
PMID:Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways. 920 14

The kinesin superfamily consists of mechanoenzymes that convert chemical energy, stored in nucleoside triphosphates, into movement along microtubules. The founding member of this protein superfamily, the so-called conventional kinesin, was only known from animal sources until the recent description of kinesin from the filamentous fungus Neurospora crassa (G. Steinberg, M. Schliwa, Mol. Biol. Cell 6, 1605-1618 (1995)). To determine whether similar motors with comparable features are common in other filamentous fungi, a kinesin from a zygomycete, Syncephalastrum racemosum, was purified. Here, the isolation and characterization of this motorenzyme is described. The purified protein consisted of a doublet at 112 kDa and 115 kDa with no additional polypeptides. This was consistent with a calculated molecular mass of approximately 240 kDa and suggests that the motor is a dimer with a more globular shape than conventional kinesin from animal sources. In gliding assays the enzyme moved microtubules at 2.5 to 3.4 microns/s and had a nucleotide specificity similar to the Neurospora kinesin motor. Peptide antibodies against conserved regions in the head and the tall domain of conventional kinesins cross-reacted with the Syncephalastrum motor. In vitro, the enzyme was able to drive the microtubule-dependent movement of vesicles isolated from Syncephalastrum racemosum, as well as Neurospora crassa, and Aspergillus nidulans. In summary, the Syncephalastrum motor has many of the unique features in common with the conventional kinesin from the ascomycete Neurospora crassa and probably shares a similar function in living hyphae.
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PMID:A kinesin-like mechanoenzyme from the zygomycete Syncephalastrum racemosum shares biochemical similarities with conventional kinesin from Neurospora crassa. 920 25

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.
J Mol Biol 1997 Aug 01
PMID:Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd. 924 92

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.
Mol Biol Cell 1997 Apr
PMID:Immunochemical analysis of kinesin light chain function. 924 47

The kinesin molecular motor "walks" processively along microtubules, touching down with alternate motor domains and transiently bridging between sites spaced 8 nm apart axially. To allow bridging, the coiled coil tail of kinesin would need to unzip a region immediately adjacent to the heads, and the tail region sequence at this point indeed contains potentially destabilising interruptions in the regular hydrophobic heptad repeat. We noticed that such interruptions are substantially absent from the coiled coil tails of Eg5, a slow kinesin homologue, and ncd, a reverse-directed homologue, and we wondered if this precluded their processivity. We measured the temperature dependence of kcat/K50% MTs, an index of the chemical processivity of a motor, the number of ATPs split per productive diffusional encounter of motor with microtubule. We found two-headed ncd (GSTMC5) and two-headed Eg5 (E437GST) constructs to be slightly if at all processive in solution over the range 4 degrees C to 30 degrees C. By contrast, two-headed kinesin constructs K401 and K430 were processive, and became substantially more so with increasing temperature. Arrhenius plots for the solution ATPase were linear for all three motors. Arrhenius plots for MT gliding assays were linear and essentially parallel for E437GST and GSTMC5 (Ea = 61 and 63 kJ mol-1) but for K430 the plot was biphasic, with a break at 17 degrees C, corresponding to a 30% reduction in Ea from 84 to 57 kJ mol-1. The data indicate that ncd and Eg5 are only slightly if at all processive, and suggest that this may be related to structural differences in their coiled coil neck regions.
J Mol Biol 1997 Oct 17
PMID:Kinetic evidence for low chemical processivity in ncd and Eg5. 936 54


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