Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intracellular transport along microtubules uses the motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein is responsible for movement to the minus ends of microtubules and the evidence indicates that dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to identify and clone the subunit polypeptides of these two complexes, in particular their light chains. Dynactin is made up of eight subunits of approximately 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide gel electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-Mr region of the gel was obtained and a candidate polypeptide identified by a screen of the databases. This polypeptide has a predicted molecular weight of 20,822 Da. Using an antibody to a different region of this protein, we demonstrate that it copurifies with microtubules and elutes from the microtubule pellet with characteristics similar to those of the dynactin complex and distinct from those of cytoplasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, but not with kinesin or cytoplasmic dynein. Together these results demonstrate that this polypeptide is the p24 subunit of dynactin. Analysis of the predicted amino acid sequence of p24 shows that it is a unique protein that has no significant similarity to known enzymes or other proteins. Structural analysis indicates that most of this protein will form an alpha-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynactin indicates that there is one molecule of p24 per dynactin complex, these characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex.
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PMID:Identification and molecular characterization of the p24 dynactin light chain. 978 90

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.
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PMID:The kinesin motor KIF3A is a component of the presynaptic ribbon in vertebrate photoreceptors. 992 Jun 66

Several X-ray crystal structures of kinesin motor domains have recently been solved at high resolution ( approximately 0.2-0.3 nm), in both their monomeric and dimeric states. They show the folding of the polypeptide chain and different arrangements of subunits in the dimer. In addition, cryo-electron microscopy and image reconstruction have revealed microtubules decorated with kinesin at intermediate resolution ( approximately 2 nm), showing the distribution and orientation of kinesin heads on the microtubule surface. The comparison of the X-ray and electron microscopy results yields a model of how monomeric motor domains bind to the microtubule but the binding of dimeric motors, their stoichiometry, or the influence of nucleotides remains a matter of debate.
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PMID:Structures of kinesin and kinesin-microtubule interactions. 1004 29

We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae. The methods examine Cin8p rapidly purified from crude yeast cell extracts. We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p. This form was biotinated in yeast cells and provided Cin8p activity in vivo. Avidin-coated glass surfaces were used to specifically bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min in the presence of ATP. Force production by Cin8p was directed toward the plus ends of microtubules. A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by >10-fold, but reduced gliding velocity by only 35%. The cin8-3 mutant form, affecting the alpha2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected. cin8-F467A cells, but not cin8-3 cells, were greatly impaired in bipolar spindle forming ability. We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation.
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PMID:Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells. 1009 42

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.
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PMID:Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana. 1019 70

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.
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PMID:Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis. 1040 68

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with </=0.1 mM NEM enhanced MC1 binding to MTs in the presence of MgATP but had no effect on MC1 binding to MTs in the presence of MgAMP-PNP. In comparison, treatment with >/=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.
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PMID:N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain. 1045 70

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.
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PMID:The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar. 1049 50

Several novel members of the kinesin superfamily, until now identified only in plants, are unique in their ability to bind calmodulin in the presence of Ca(2+). Here, we identify the first such kinesin in an animal system. Sequence analysis of this new motor, called kinesin-C, predicts that it is a large carboxy-terminal kinesin, 1624 amino acid residues in length, with a predicted molecular mass of 181 kDa. Kinesin-C is predicted to contain a kinesin motor domain at its carboxy terminus, linked to a segment of alpha-helical coiled-coil 950 amino acid residues long, ending with an amino-terminal proline-rich tail domain. A putative calmodulin-binding domain resides at the extreme carboxy terminus of the motor polypeptide, and recombinant kinesin-C binds to a calmodulin-affinity column in a Ca(2+)-dependent fashion. The presence of this novel calmodulin-binding motor in sea urchin embryos suggests that it plays a critical role in Ca(2+)-dependent events during early sea urchin development.
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PMID:Identification of kinesin-C, a calmodulin-binding carboxy-terminal kinesin in animal (Strongylocentrotus purpuratus) cells. 1055 23

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.
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PMID:Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes. 1100 43


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