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)

We report here the complete sequence of the gamma dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The gamma dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high alpha-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the gamma dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.
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PMID:Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein. 751 41

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992) J. Cell Sci. 101, 303-313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, 'LAGSE' and, HIPYRESKLT, 'HIPYR') reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, approximately 110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the '97 kDa' proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotide-dependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.
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PMID:Kinesin-related proteins in eukaryotic flagella. 796 96

Affinity-purified polyclonal antibodies raised against the conserved motor domain of Drosophila kinesin (alpha HD) recognized a 110 kDa component of the Chlamydomonas flagellar axoneme. Whole-mount immunogold labeling of splayed axonemes showed the striking localization of this antigen along one of the two microtubules of the central pair apparatus. Interestingly, the alpha HD antigen was also localized along the central axis of mutant axonemes lacking the central pair microtubules. These results suggest that a 110 kDa kinesin-related protein is a structural component of the flagellar central pair apparatus and that it is correctly targeted even in the absence of the central pair microtubules.
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PMID:Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum. 796 97

Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutations cause temperature-sensitive defects in flagellar assembly. We present the molecular analysis of a gene which maps to fla10 and functionally rescues the fla10 phenotype. Nucleotide sequencing revealed that the gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicted KHP1 protein, like kinesin heavy chain, has an amino-terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-terminal tail. Comparison to other kinesin superfamily members indicated striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, the KHP1 mRNA accumulated after cell division, as did flagellar dynein mRNAs. KHP1 mRNA levels also increased following deflagellation. Polyclonal antibodies detected KHP1 protein in Western blots of purified flagella and axonemes. The protein was partially released from axonemes with ATP treatment, but not with AMP-PNP. Western blot analysis of axonemes from various motility mutants suggested that KHP1 is not a component of radial spokes, dynein arms, or the central pair complex. The quantity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage group mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by transformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-related protein in flagellar assembly and maintenance.
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PMID:The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. 802 76

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.
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PMID:A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum. 820 60

Microtubules nucleated by sea urchin sperm-tail axonemes have polar ends that differ both functionally and structurally but cannot be distinguished from one another when viewed by light microscopy. Ambiguity and circularity surround any classification of microtubule polarity by conventional methods. Chlamydomonas flagellar axonemal pieces have distinct morphological differences at their plus- and minus-ends, and microtubules nucleated from these pieces can be distinguished as plus- or minus-ended based on the morphological differences present in the Chlamydomonas flagellar axonemal pieces. Plus- and minus-ended microtubules were polymerized in this fashion and analyzed for differences in growth rates, shortening rates, and frequencies of transitions. The results were in good agreement with similar data generated by the more time-consuming and difficult use of kinesin-coated beads (R. J. Kowalski, and R. C. Williams, Jr. (1993) Cell Motil. Cytoskeleton 26, 282-290) to determine microtubule polarity. This is a relatively simple and effective method for determining the polarity of microtubules in vitro by video-enhanced differential-interference contrast light microscopy.
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PMID:Determination of microtubule polarity in vitro by the use of video-enhanced differential-interference contrast light microscopy and Chlamydomonas flagellar axonemal pieces. 860 Aug 30

Sequence comparisons with the Mr 8,000 light chain from Chlamydomonas outer arm dynein revealed the presence of highly conserved homologues (up to 90% identity) in the expressed sequence tag data base (King, S. M. & Patel-King, R. S. (1995a) J. Biol. Chem. 270, 11445-11452). Several of these homologous sequences were derived from organisms and/or tissues that lack motile cilia/flagella, suggesting that these proteins may function in the cytoplasm. In Drosophila, lack of the homologous protein results in embryonic lethality (Dick, T., Ray, K., Salz, H. K. & Chia, W.(1996) Mol. Cell. Biol., 16, 1966-1977). Fractionation of mammalian brain homogenates reveals three distinct cytosolic pools of the homologous protein, one of which specifically copurifies with cytoplasmic dynein following both ATP-sensitive microtubule affinity/sucrose density gradient centrifugation and immunoprecipitation with a monoclonal antibody specific for the 74-kDa intermediate chain (IC74). Quantitative densitometry indicates that there is one copy of the Mr 8,000 polypeptide per IC74. Dual channel confocal immunofluorescent microscopy revealed that the Mr 8,000 protein is significantly colocalized with cytoplasmic dynein but not with kinesin in punctate structures (many of which are associated with microtubules) within mammalian oligodendrocytes. Thus, it appears that flagellar outer arm and brain cytoplasmic dyneins share a highly conserved light chain polypeptide that, at least in Drosophila, is essential for viability.
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PMID:Brain cytoplasmic and flagellar outer arm dyneins share a highly conserved Mr 8,000 light chain. 870 22

Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.
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PMID:Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors. 871 80

We previously reported that the FLA10 locus on the uni linkage group of Chlamydomonas encodes a kinesin homologous protein, KHP1. The fla10 phenotype, which is a temperature-sensitive defect for flagellar assembly and maintenance, is rescued by transformation with the wild-type KHP1 gene. In the present study we identify the molecular defect associated with the fla10 mutation and examine the subcellular localization of KHP1 throughout the cell cycle. The mutation in the fla10-1 allele consists of a C to A transversion, which alters amino acid 329 in the motor domain of KHP1. This residue and the sequence of the carboxy-terminal third of the motor domain in which it is located are highly conserved throughout eukaryotic evolution in a subfamily of kinesin-related proteins from mouse (KIF3), sea urchin (KRP85/95), Xenopus (XKLP3), and Drosophila (KLP68D). These data suggest a conserved function for this family of proteins. Immunofluorescence studies reveal that: (1) in interphase cells KHP1 is associated with basal bodies and with the proximal portion of the flagella; (2) in cells undergoing flagellar regeneration KHP1 occurs in punctate structures that extend to the tip of the developing axoneme; and (3) in dividing cells KHP1 remains associated with centrioles throughout mitosis and localizes to the mitotic spindle. KHP1 is the first kinesin homologous protein to be found in association with basal bodies and centrioles throughout the cell cycle. These observations provide evidence for a direct role of basal bodies in the process of flagellar development, which we propose is based on KHP1 acting as a transporter of flagellar components from the basal bodies out to the distal site of assembly. The localization of KHP1 in mitosis suggests that this protein may play an analogous role in the centriole-based assembly of the mitotic spindle.
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PMID:The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles. 890

Proteins necessary for maintenance and function of eukaryotic flagella are synthesized in the cell body. Transport of the inner dynein arm subunit p28(IDA4) in Chlamydomonas flagella requires the activity of the kinesin KHP1(FLA10), a protein inactive at restrictive temperature in fla10, a temperature-dependent mutant of flagellar assembly. To identify other molecules involved in active transport of inner dynein arms within flagella we searched for polypeptides of the cytoplasmic matrix of flagella that fulfill two conditions: they bind to p28 and require the activity of KHP1 to be present in flagella. We found that the cytoplasmic matrix of flagella contains a previously unidentified "17S" complex of at least 13 polypeptides that in part is associated with p28. The 17S complex is present at permissive but not at restrictive temperature in fla10 flagella. It also turns over in the cytoplasmic matrix more frequently than dynein arms within the axoneme. This evidence suggests that the 17S complex transports precursors of inner dynein arms within flagella.
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PMID:Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella. 911 11


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