Abstract
Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
Purchase on Springer Link
Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Vale, R. D. in Guidebook to Cytoskeletal and Motor Proteins 2nd edn (eds Kreis, T. E. & Vale, R. D.) 398–402 (Oxford Univ. Press, Oxford, 1999).
Hirokawa, N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519–526 (1998).
Liao, G. & Gundersen, G. Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. J. Biol. Chem. 273, 9797–9803 (1998).
Prahlad, V., Yoon, M., Moir, R. D., Vale, R.D. & Goldman, R. D. Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J. Cell Biol. 143, 159–170 (1998).
Bloom, G. S., Wagner, M. C., Pfister, K. K. & Brady, S. T. Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. Biochemistry 27, 3409–3416 (1988).
Kuznetsov, S. A. et al. The quaternary structure of bovine brain kinesin. EMBO J. 7, 353–356 (1988).
Yang, J. T., Laymon, R. A. & Goldstein, L. S. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56, 879–889 (1989).
Henningsen, U. & Schliwa, M. Reversal in the direction of movement of a molecular motor. Nature 389, 93–96 (1997).
Endow, S. A. & Waligora, K. W. Determinants of kinesin motor polarity. Science 281, 1200–1202 (1998).
Case, R. B., Pierce, D. W., Hom-Booher, N., Hart, C. L. & Vale, R. D. The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain. Cell 90, 959–966 (1997).
Hirokawa, N. et al. Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell 56, 867–878 (1989).
Hisanaga, S. et al. The molecular structure of adrenal medulla kinesin. Cell Motil. Cytoskeleton 12, 264–272 (1989).
Diefenbach, R. J., Mackay, J. P., Armati, P. J. & Cunningham, A. L. The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain. Biochemistry 37, 16663–16670 (1998).
Verhey, K. J. et al. Light-chain dependent regulation of kinesin’s interaction with microtubules. J. Cell Biol. 143, 1053–1066 (1998).
Skoufias, D., Cole, D. G., Wedaman, K. P. & Scholey, J. M. The carboxyl-terminal domain of kinesin heavy chain is important for membrane binding. J. Biol. Chem. 269, 1477–1485 (1994).
Hollenbeck, P. J. The distribution, abundance and subcellular localization of kinesin. J. Biol. Chem. 108, 2335–2342 (1989).
Niclas, J., Navone, F., Hom-Booher, N. & Vale, R. D. Cloning and localization of a conventional kinesin motor expressed exclusively in neurons. Neuron 12, 1059–1072 (1994).
Hackney, D. D., Levitt, J. D. & Suhan, J. Kinesin undergoes a 9 S to 6 S conformational transition. J. Biol. Chem. 267, 8696–8701 (1992).
Jiang, M. Y. & Sheetz, M. P. Cargo-activated ATPase activity of kinesin. Biophys. J. 68 (suppl.), 283–285 (1995).
Moraga, D. E. & Murphy, D. B. Kinesin is ‘‘inactive’’ unless bound to a solid support. Mol. Biol. Cell Abstr. 8, 258 (1997).
Kuznetsov, S. A., Vaisberg, Y. A., Rothwell, S. W., Murphy, D. B. & Gelfand, V. I. Isolation of a 45-kDA fragment from the kinesin heavy chain with enhanced ATPase and microtubule-binding activities. J. Biol. Chem. 264, 589–595 (1989).
Stock, M. et al. Formation of the compact conformer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity. J. Biol. Chem. 274, 14617–14623 (1999).
Hackney, D. D., Levitt, J. D. & Wagner, D. D. Characterization of α2β2 and α2 forms of kinesin. Biochem. Biophys. Res. Commun. 174, 810–815 (1991).
Vale, R. D., Reese, T. S. & Sheetz, M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50 (1985).
Cohn, S. A., Ingold, A. L. & Scholey, J. M. Quantitative analysis of sea urchin egg kinesin-driven microtubule motility. J. Biol. Chem. 264, 4290–4297 (1989).
Yang, J. T., Saxton, W. M., Stewart, R. J., Raff, E. C. & Goldstein, L. S. Evidence that the head of kinesin is sufficient for force generation and motility in vitro. Science 249, 42–47 (1990).
Berliner, E., Young, E. C., Anderson, K., Mahtani, H. & Gelles, J. Failure of a single-headed kinesin to track parallel to microtubule protofilaments. Nature 373, 718–721 (1995).
Vale, R. D. et al. Direct observation of single kinesin molecules moving along microtubules. Nature 380, 451–453 (1996).
Woehlke, G. et al. Microtubule interaction site of the kinesin motor. Cell 90, 207–216 (1997).
Wagner, M. C., Pfister, K. K., Bloom, G. S. & Brady, S. T. Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties. Cell Motil. Cytoskeleton 12, 195–215 (1989).
Navone, F. et al. Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J. Cell Biol. 117, 1263–1275 (1992).
Andrews, S. B., Gallant, P. E., Leapman, R. D., Schnapp, B. J. & Reese, T. S. Single kinesin molecules crossbridge microtubules in vitro. Proc. Natl Acad. Sci. USA 90, 6503–6507 (1993).
Kozielski, F. et al. The crystal structure of dimeric kinesin and implications for microtubule-dependent motility. Cell 91, 985–994 (1997).
Tripet, B., Vale, R. D. & Hodges, R. S. Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides: implications for motor activity. J. Biol. Chem. 272, 8946–8956 (1997).
Romberg, L., Pierce, D. W. & Vale, R. D. Role of the kinesin neck region in processive microtubule-based motility. J. Cell Biol. 140, 1407–1416 (1998).
Grummt, M. et al. Importance of a flexible hinge near the motor domain in kinesin-driven motility. EMBO J. 17, 5536–5542 (1998).
Vale, R. D. & Fletterick, R. J. The design plan of kinesin motors. Annu. Rev. Cell Dev. Biol. 13, 745–777 (1997).
Pfister, K. K., Wagner, M. C., Stenoien, D. L., Brady, S. T. & Bloom, G. S. Monoclonal antibodies to kinesin heavy and lights chains stain vesicle-like structures, but not microtubules, in cultured cells. J. Cell Biol. 108, 1453–1463 (1989).
Bi, G. Q. et al. Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis. J. Cell Biol. 138, 999–1008 (1997).
Lee, K. D. & Hollenbeck, P. J. Phosphorylation of kinesin in vivo correlates with organelle association and neurite outgrowth. J. Biol. Chem. 270, 5600–5605 (1995).
Matthies, H. J., Miller, R. J. & Palfrey, H. C. Calmodulin binding to and cAMP-dependent phosphorylation of kinesin light chains modulate kinesin ATPase activity. J. Biol. Chem. 268, 11176–11187 (1993).
McIlvain, J. M., Burkhardt, J. K., Hamm-Alvarez, S., Argon, Y. & Sheetz, M. P. Regulation of kinesin activity by phosphorylation of kinesin-associated proteins. J. Biol. Chem. 269, 19176–19182 (1994).
Lindesmith, L., McIlvain, J. M., Argon, Y. & Sheetz, M. P. Phosphotransferases associated with the regulation of kinesin motor activity. J. Biol. Chem. 272, 22929–22933 (1997).
Hollenbeck, P. J. Phosphorylation of neuronal kinesin heavy and light chains in vivo. J. Neurochem. 60, 2265–2275 (1993).
Cabeza-Arvelaiz, Y. et al. Cloning and genetic characterization of the human kinesin light chain (KLC) gene. DNA Cell Biol. 12, 881–892 (1993).
Kodama, T., Fukui, K. & Kometani, K. The initial phosphate burst in ATP hydrolysis by myosin and subfragment 1 as studied by a modified Malachite Green method for determination of organic phosphate. J. Biochem. 99, 1465–1472 (1986).
Pierce, D. W. & Vale, R. D. Assaying processive movement of kinesin by fluorescence microscopy. Methods Enzymol. 298, 154–171 (1998).
Gibbons, I. R. & Fronk, E. A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J. Biol. Chem. 254, 187–196 (1979).
Acknowledgements
We acknowlege the members of the Vale laboratory for their help in many aspects of this work. We thank J. Hartman, S. Hopkins, D. Pierce, A. Rudner and K. Thorn for insightful discussion, experimental help, and assistance with the manuscript; C. Hart and J. Ubersax for preparing the K560 (neck mut.) clone; L. Lachman and Y. Cabeza-Alvelaiz for providing the kinesin light chain clone and technical advice. D.S.F. is supported by UCSF Cell Biology Training Grant number T32 GM08120.
Correspondence and requests for materials should be addressed to R.D.V. The protein sequences for human ubiquitous kinesin heavy chain and light chain have been deposited at the Protein DataBank under accession numbers U06698 and L04733, respectively.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Friedman, D., Vale, R. Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain. Nat Cell Biol 1, 293–297 (1999). https://doi.org/10.1038/13008
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/13008
This article is cited by
-
Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs
BMC Biology (2022)
-
The architecture of kinesin-3 KLP-6 reveals a multilevel-lockdown mechanism for autoinhibition
Nature Communications (2022)
-
Kinesin-1 activity recorded in living cells with a precipitating dye
Nature Communications (2021)
-
Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments
Nature Communications (2020)
-
JIP1 and JIP3 cooperate to mediate TrkB anterograde axonal transport by activating kinesin-1
Cellular and Molecular Life Sciences (2017)