Analysis of Calcium/Calmodulin Regulation of a Plant Kinesin Using Co-Sedimentation and ATPase Assays

  • Anireddy S. N. Reddy
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)


Kinesins, a superfamily of microtubule motor proteins, are implicated in regulating a number of fundamental cellular and developmental processes including intracellular transport of vesicles and organelles, mitotic and meiotic spindle formation and elongation, chromosome segregation, germplasm aggregation, microtubule (MT) organization and dynamics, and intraflagellar transport. Analysis of all the completed genomes of eukaryotes has revealed that Arabidopsis, a flowering plant, has more kinesins than any other organism. Although a complete inventory of kinesins in a number of organisms has been reported, the function and regulation of kinesins in general and plant kinesins in particular are poorly understood. In our screen of an expression library with a labeled calmodulin, we isolated a novel plant kinesin (kinesin-like calmodulin-binding protein, KCBP) from plants, which interacts with calmodulin in a calcium-dependent manner. This chapter describes the methods used in elucidating the regulation of this motor protein by calcium/calmodulin.

Key Words

calcium calmodulin KCBP, co-sedimentation assay ATPase assay kinesin motor protein microtubule (MT) 


  1. 1.
    Reddy, A.S. (2001) Calcium: silver bullet in signaling. Plant Sci. 160, 381–404.CrossRefPubMedGoogle Scholar
  2. 2.
    Reddy, V.S. and Reddy, A.S. (2004) Proteomics of calcium-signaling components in plants. Phytochemistry 65, 1745–1776.CrossRefPubMedGoogle Scholar
  3. 3.
    Day, I.S., Reddy, V.S., Ali, G.S., and Reddy, A.S.N. (2002) Analysis of EF-hand-containing proteins in Arabidopsis. Genome Biol. 3, 56.1–56.24.Google Scholar
  4. 4.
    Reddy, V.S., Ali, G.S., and Reddy, A.S.N. (2002) Genes encoding calmodulin-binding proteins in the Arabidopsis genome. J. Biol. Chem. 277, 9840–9852.CrossRefPubMedGoogle Scholar
  5. 5.
    Reddy, A.S.N., Safadi, F., Narasimhulu, S.B., Golovkin, M., and Hu, X. (1996) A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain. J. Biol. Chem. 271, 7052–7060.CrossRefPubMedGoogle Scholar
  6. 6.
    Abdel-Ghany, S.E., Day, I.S., Simmons, M.P., Kugrens, P., and Reddy, A.S. (2005) Origin and evolution of kinesin-like calmodulin-binding protein. Plant Physiol. 138, 1711–1722.CrossRefPubMedGoogle Scholar
  7. 7.
    Narasimhulu, S.B., and Reddy, A.S.N. (1998) Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin-binding protein. Plant Cell 10, 957–965.CrossRefPubMedGoogle Scholar
  8. 8.
    Reddy, V.S., Day, I.S., Thomas, T., and Reddy, A.S. (2004) KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell 16, 185–200.CrossRefPubMedGoogle Scholar
  9. 9.
    Reddy, V.S., and Reddy, A.S.N. (2002) The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-CaM regulation to animal plus-and minus-end kinesins. J. Biol. Chem. 277, 48058–48065.CrossRefPubMedGoogle Scholar
  10. 10.
    Narasimhulu, S.B., Kao, Y.-L., and Reddy, A.S.N. (1997) Interaction of Arabidopsis kinesin-like calmodulin-binding protein with tubulin subunits: modulation by Ca2+-calmodulin. Plant J. 12, 1139–1149.CrossRefPubMedGoogle Scholar
  11. 11.
    Liao, B., Gawienowski, M.C., and Zielinski, R.E. (1996) Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms. Arch. Biochem. Biophys. 327, 53–60.CrossRefPubMedGoogle Scholar
  12. 12.
    Vale, R.D., Reese, T.S., and Sheetz, M.P. (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50.CrossRefPubMedGoogle Scholar
  13. 13.
    Yang, J.T., Laymon, R.A., and Goldstein, L.S.B. (1989) A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56, 879–889.CrossRefPubMedGoogle Scholar
  14. 14.
    Chandra, R., and Endow, S.A. (1993) In: Methods in Cell Biology (Wilson, L. and Matsudaira, P., eds.), vol. 39, pp. 115–128. Academic Press, New York.Google Scholar
  15. 15.
    Hackney, D.D. and Jiang, W. (2001) Assays for kinesin microtubule-stimulated ATPase activity. Methods Mol. Biol. 164, 65–71.PubMedGoogle Scholar
  16. 16.
    Penney, C.L. and Bolger, G. (1978) A simple microassay for inorganic phosphate. II. Anal. Biochem. 89, 297–303.CrossRefPubMedGoogle Scholar
  17. 17.
    Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., and Candia, O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95–97.CrossRefPubMedGoogle Scholar
  18. 18.
    Reddy, V.S. and Reddy, A.S.N. (1999) A plant calmodulin-binding motor is part kinesin and part myosin. Bioinformatics 15, 1055–1057.CrossRefPubMedGoogle Scholar
  19. 19.
    Reddy, V.S., Safadi, F., Zielinski, R.E., and Reddy, A.S.N. (1999) Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis. J. Biol. Chem. 274, 31727–31733.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Anireddy S. N. Reddy
    • 1
  1. 1.Department of Biology and Program in Cell and Molecular BiologyColorado State UniversityFort Collins

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