Abstract
Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194- 200), indicating that HSET might represent a human homologue of the CHO2 antigen.
Full Text
The Full Text of this article is available as a PDF (2.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aizawa H., Sekine Y., Takemura R., Zhang Z., Nangaku M., Hirokawa N. Kinesin family in murine central nervous system. J Cell Biol. 1992 Dec;119(5):1287–1296. doi: 10.1083/jcb.119.5.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ando A., Kikuti Y. Y., Kawata H., Okamoto N., Imai T., Eki T., Yokoyama K., Soeda E., Ikemura T., Abe K. Cloning of a new kinesin-related gene located at the centromeric end of the human MHC region. Immunogenetics. 1994;39(3):194–200. doi: 10.1007/BF00241260. [DOI] [PubMed] [Google Scholar]
- Bernstein M., Beech P. L., Katz S. G., Rosenbaum J. L. A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum. J Cell Biol. 1994 Jun;125(6):1313–1326. doi: 10.1083/jcb.125.6.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chandra R., Salmon E. D., Erickson H. P., Lockhart A., Endow S. A. Structural and functional domains of the Drosophila ncd microtubule motor protein. J Biol Chem. 1993 Apr 25;268(12):9005–9013. [PubMed] [Google Scholar]
- Cole D. G., Cande W. Z., Baskin R. J., Skoufias D. A., Hogan C. J., Scholey J. M. Isolation of a sea urchin egg kinesin-related protein using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):291–301. doi: 10.1242/jcs.101.2.291. [DOI] [PubMed] [Google Scholar]
- Endow S. A., Chandra R., Komma D. J., Yamamoto A. H., Salmon E. D. Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis. J Cell Sci. 1994 Apr;107(Pt 4):859–867. doi: 10.1242/jcs.107.4.859. [DOI] [PubMed] [Google Scholar]
- Endow S. A., Henikoff S., Soler-Niedziela L. Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin. Nature. 1990 May 3;345(6270):81–83. doi: 10.1038/345081a0. [DOI] [PubMed] [Google Scholar]
- Endow S. A., Kang S. J., Satterwhite L. L., Rose M. D., Skeen V. P., Salmon E. D. Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends. EMBO J. 1994 Jun 1;13(11):2708–2713. doi: 10.1002/j.1460-2075.1994.tb06561.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Enos A. P., Morris N. R. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell. 1990 Mar 23;60(6):1019–1027. doi: 10.1016/0092-8674(90)90350-n. [DOI] [PubMed] [Google Scholar]
- Fox L. A., Sawin K. E., Sale W. S. Kinesin-related proteins in eukaryotic flagella. J Cell Sci. 1994 Jun;107(Pt 6):1545–1550. doi: 10.1242/jcs.107.6.1545. [DOI] [PubMed] [Google Scholar]
- Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gelfand V. I., Scholey J. M. Cell biology. Every motion has its motor. Nature. 1992 Oct 8;359(6395):480–482. doi: 10.1038/359480a0. [DOI] [PubMed] [Google Scholar]
- Goldstein L. S. With apologies to scheherazade: tails of 1001 kinesin motors. Annu Rev Genet. 1993;27:319–351. doi: 10.1146/annurev.ge.27.120193.001535. [DOI] [PubMed] [Google Scholar]
- Goodson H. V., Kang S. J., Endow S. A. Molecular phylogeny of the kinesin family of microtubule motor proteins. J Cell Sci. 1994 Jul;107(Pt 7):1875–1884. doi: 10.1242/jcs.107.7.1875. [DOI] [PubMed] [Google Scholar]
- Hagan I., Yanagida M. Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature. 1990 Oct 11;347(6293):563–566. doi: 10.1038/347563a0. [DOI] [PubMed] [Google Scholar]
- Harada Y., Sakurada K., Aoki T., Thomas D. D., Yanagida T. Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. J Mol Biol. 1990 Nov 5;216(1):49–68. doi: 10.1016/S0022-2836(05)80060-9. [DOI] [PubMed] [Google Scholar]
- Heck M. M., Pereira A., Pesavento P., Yannoni Y., Spradling A. C., Goldstein L. S. The kinesin-like protein KLP61F is essential for mitosis in Drosophila. J Cell Biol. 1993 Nov;123(3):665–679. doi: 10.1083/jcb.123.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoyt M. A., He L., Loo K. K., Saunders W. S. Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly. J Cell Biol. 1992 Jul;118(1):109–120. doi: 10.1083/jcb.118.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hyman A. A. Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence. J Cell Sci Suppl. 1991;14:125–127. doi: 10.1242/jcs.1991.supplement_14.25. [DOI] [PubMed] [Google Scholar]
- Hyman A., Drechsel D., Kellogg D., Salser S., Sawin K., Steffen P., Wordeman L., Mitchison T. Preparation of modified tubulins. Methods Enzymol. 1991;196:478–485. doi: 10.1016/0076-6879(91)96041-o. [DOI] [PubMed] [Google Scholar]
- Johnson K. A., Haas M. A., Rosenbaum J. L. Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum. J Cell Sci. 1994 Jun;107(Pt 6):1551–1556. doi: 10.1242/jcs.107.6.1551. [DOI] [PubMed] [Google Scholar]
- Kimble M., Kuriyama R. Functional components of microtubule-organizing centers. Int Rev Cytol. 1992;136:1–50. doi: 10.1016/s0074-7696(08)62049-5. [DOI] [PubMed] [Google Scholar]
- Kondo S., Sato-Yoshitake R., Noda Y., Aizawa H., Nakata T., Matsuura Y., Hirokawa N. KIF3A is a new microtubule-based anterograde motor in the nerve axon. J Cell Biol. 1994 Jun;125(5):1095–1107. doi: 10.1083/jcb.125.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuriyama R., Dragas-Granoic S., Maekawa T., Vassilev A., Khodjakov A., Kobayashi H. Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells. J Cell Sci. 1994 Dec;107(Pt 12):3485–3499. doi: 10.1242/jcs.107.12.3485. [DOI] [PubMed] [Google Scholar]
- Kuriyama R., Keryer G., Borisy G. G. The mitotic spindle of Chinese hamster ovary cells isolated in taxol-containing medium. J Cell Sci. 1984 Mar;66:265–275. doi: 10.1242/jcs.66.1.265. [DOI] [PubMed] [Google Scholar]
- Kuriyama R., Nislow C. Molecular components of the mitotic spindle. Bioessays. 1992 Feb;14(2):81–88. doi: 10.1002/bies.950140203. [DOI] [PubMed] [Google Scholar]
- Maekawa T., Kuriyama R. Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA. J Cell Sci. 1993 Jun;105(Pt 2):589–600. doi: 10.1242/jcs.105.2.589. [DOI] [PubMed] [Google Scholar]
- McDonald H. B., Goldstein L. S. Identification and characterization of a gene encoding a kinesin-like protein in Drosophila. Cell. 1990 Jun 15;61(6):991–1000. doi: 10.1016/0092-8674(90)90064-l. [DOI] [PubMed] [Google Scholar]
- McDonald H. B., Stewart R. J., Goldstein L. S. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell. 1990 Dec 21;63(6):1159–1165. doi: 10.1016/0092-8674(90)90412-8. [DOI] [PubMed] [Google Scholar]
- McIntosh J. R., Pfarr C. M. Mitotic motors. J Cell Biol. 1991 Nov;115(3):577–585. doi: 10.1083/jcb.115.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meluh P. B., Rose M. D. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. doi: 10.1016/0092-8674(90)90351-e. [DOI] [PubMed] [Google Scholar]
- Mitsui H., Yamaguchi-Shinozaki K., Shinozaki K., Nishikawa K., Takahashi H. Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA. Mol Gen Genet. 1993 Apr;238(3):362–368. doi: 10.1007/BF00291995. [DOI] [PubMed] [Google Scholar]
- Nislow C., Lombillo V. A., Kuriyama R., McIntosh J. R. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature. 1992 Oct 8;359(6395):543–547. doi: 10.1038/359543a0. [DOI] [PubMed] [Google Scholar]
- O'Connell M. J., Meluh P. B., Rose M. D., Morris N. R. Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans. J Cell Biol. 1993 Jan;120(1):153–162. doi: 10.1083/jcb.120.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Page B. D., Satterwhite L. L., Rose M. D., Snyder M. Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein. J Cell Biol. 1994 Feb;124(4):507–519. doi: 10.1083/jcb.124.4.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pesavento P. A., Stewart R. J., Goldstein L. S. Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport. J Cell Biol. 1994 Nov;127(4):1041–1048. doi: 10.1083/jcb.127.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roof D. M., Meluh P. B., Rose M. D. Kinesin-related proteins required for assembly of the mitotic spindle. J Cell Biol. 1992 Jul;118(1):95–108. doi: 10.1083/jcb.118.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saunders W. S., Hoyt M. A. Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell. 1992 Aug 7;70(3):451–458. doi: 10.1016/0092-8674(92)90169-d. [DOI] [PubMed] [Google Scholar]
- Sawin K. E., Endow S. A. Meiosis, mitosis and microtubule motors. Bioessays. 1993 Jun;15(6):399–407. doi: 10.1002/bies.950150606. [DOI] [PubMed] [Google Scholar]
- Sawin K. E., Mitchison T. J., Wordeman L. G. Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):303–313. doi: 10.1242/jcs.101.2.303. [DOI] [PubMed] [Google Scholar]
- Sellitto C., Kimble M., Kuriyama R. Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus-associated bodies from dictyostelium. Cell Motil Cytoskeleton. 1992;22(1):7–24. doi: 10.1002/cm.970220103. [DOI] [PubMed] [Google Scholar]
- Sellitto C., Kuriyama R. Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells. J Cell Biol. 1988 Feb;106(2):431–439. doi: 10.1083/jcb.106.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Vale R. D., Goldstein L. S. One motor, many tails: an expanding repertoire of force-generating enzymes. Cell. 1990 Mar 23;60(6):883–885. doi: 10.1016/0092-8674(90)90334-b. [DOI] [PubMed] [Google Scholar]
- Vassilev A., Kimble M., Silflow C. D., LaVoie M., Kuriyama R. Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence. J Cell Sci. 1995 Mar;108(Pt 3):1083–1092. doi: 10.1242/jcs.108.3.1083. [DOI] [PubMed] [Google Scholar]
- Walker R. A., Salmon E. D., Endow S. A. The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature. 1990 Oct 25;347(6295):780–782. doi: 10.1038/347780a0. [DOI] [PubMed] [Google Scholar]
- Wordeman L., Mitchison T. J. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J Cell Biol. 1995 Jan;128(1-2):95–104. doi: 10.1083/jcb.128.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yen T. J., Compton D. A., Wise D., Zinkowski R. P., Brinkley B. R., Earnshaw W. C., Cleveland D. W. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1991 May;10(5):1245–1254. doi: 10.1002/j.1460-2075.1991.tb08066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yen T. J., Li G., Schaar B. T., Szilak I., Cleveland D. W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992 Oct 8;359(6395):536–539. doi: 10.1038/359536a0. [DOI] [PubMed] [Google Scholar]
- Yeom Y. I., Abe K., Bennett D., Artzt K. Testis-/embryo-expressed genes are clustered in the mouse H-2K region. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):773–777. doi: 10.1073/pnas.89.2.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang P., Knowles B. A., Goldstein L. S., Hawley R. S. A kinesin-like protein required for distributive chromosome segregation in Drosophila. Cell. 1990 Sep 21;62(6):1053–1062. doi: 10.1016/0092-8674(90)90383-p. [DOI] [PubMed] [Google Scholar]