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. 2000 Apr;78(4):1955–1964. doi: 10.1016/S0006-3495(00)76743-9

The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity.

Z Wang 1, M P Sheetz 1
PMCID: PMC1300788  PMID: 10733974

Abstract

In motor movement on microtubules, the anionic C-terminal of tubulin has been implicated as a significant factor. Our digital analyses of movements of cytoplasmic dynein- and kinesin-coated beads on microtubules have revealed dramatic changes when the C-terminal region (2-4-kDa fragment) of tubulin was cleaved by limited subtilisin digestion of assembled microtubules. For both motors, bead binding to microtubules was decreased threefold, bead run length was decreased over fourfold, and there was a dramatic 20-fold decrease in diffusional movements of cytoplasmic dynein beads on microtubules (even with low motor concentrations where the level of bead motile activity was linear with motor concentration). The velocity of active bead movements on microtubules was unchanged for cytoplasmic dynein and slightly decreased for kinesin. There was also a decrease in the frequency of bead movements without a change in velocity when the ionic strength was raised. However, with high ionic strength there was not a decrease in run length or any selective inhibition of the diffusional movement. The C-terminal region of tubulin increased motor run length (processivity) by inhibiting "detachment" but without affecting velocity. Because the major motor binding sites of microtubules are not on the C-terminal tail of tubulin (), we suggest that the changes are the result of the compromise of a weakly attached state that is the lowest affinity step in both motors' ATPase cycles and is not rate limiting.

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Selected References

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  1. Berliner E., Young E. C., Anderson K., Mahtani H. K., Gelles J. Failure of a single-headed kinesin to track parallel to microtubule protofilaments. Nature. 1995 Feb 23;373(6516):718–721. doi: 10.1038/373718a0. [DOI] [PubMed] [Google Scholar]
  2. Bhattacharyya B., Sackett D. L., Wolff J. Tubulin, hybrid dimers, and tubulin S. Stepwise charge reduction and polymerization. J Biol Chem. 1985 Aug 25;260(18):10208–10216. [PubMed] [Google Scholar]
  3. Block S. M., Goldstein L. S., Schnapp B. J. Bead movement by single kinesin molecules studied with optical tweezers. Nature. 1990 Nov 22;348(6299):348–352. doi: 10.1038/348348a0. [DOI] [PubMed] [Google Scholar]
  4. Chandra R., Endow S. A., Salmon E. D. An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays. J Cell Sci. 1993 Mar;104(Pt 3):899–906. doi: 10.1242/jcs.104.3.899. [DOI] [PubMed] [Google Scholar]
  5. Coppin C. M., Finer J. T., Spudich J. A., Vale R. D. Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1913–1917. doi: 10.1073/pnas.93.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coppin C. M., Pierce D. W., Hsu L., Vale R. D. The load dependence of kinesin's mechanical cycle. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8539–8544. doi: 10.1073/pnas.94.16.8539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gelles J., Schnapp B. J., Sheetz M. P. Tracking kinesin-driven movements with nanometre-scale precision. Nature. 1988 Feb 4;331(6155):450–453. doi: 10.1038/331450a0. [DOI] [PubMed] [Google Scholar]
  8. Hackney D. D. Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains. Nature. 1995 Oct 5;377(6548):448–450. doi: 10.1038/377448a0. [DOI] [PubMed] [Google Scholar]
  9. Hackney D. D. The kinetic cycles of myosin, kinesin, and dynein. Annu Rev Physiol. 1996;58:731–750. doi: 10.1146/annurev.ph.58.030196.003503. [DOI] [PubMed] [Google Scholar]
  10. Hagiwara H., Yorifuji H., Sato-Yoshitake R., Hirokawa N. Competition between motor molecules (kinesin and cytoplasmic dynein) and fibrous microtubule-associated proteins in binding to microtubules. J Biol Chem. 1994 Feb 4;269(5):3581–3589. [PubMed] [Google Scholar]
  11. Hancock W. O., Howard J. Processivity of the motor protein kinesin requires two heads. J Cell Biol. 1998 Mar 23;140(6):1395–1405. doi: 10.1083/jcb.140.6.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heins S., Song Y. H., Wille H., Mandelkow E., Mandelkow E. M. Effect of MAP2, MAP2c, and tau on kinesin-dependent microtubule motility. J Cell Sci Suppl. 1991;14:121–124. doi: 10.1242/jcs.1991.supplement_14.24. [DOI] [PubMed] [Google Scholar]
  13. Hirose K., Fan J., Amos L. A. Re-examination of the polarity of microtubules and sheets decorated with kinesin motor domain. J Mol Biol. 1995 Aug 18;251(3):329–333. doi: 10.1006/jmbi.1995.0437. [DOI] [PubMed] [Google Scholar]
  14. Hirose K., Lockhart A., Cross R. A., Amos L. A. Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9539–9544. doi: 10.1073/pnas.93.18.9539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoenger A., Milligan R. A. Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J Mol Biol. 1997 Feb 7;265(5):553–564. doi: 10.1006/jmbi.1996.0757. [DOI] [PubMed] [Google Scholar]
  16. Hunt A. J., Gittes F., Howard J. The force exerted by a single kinesin molecule against a viscous load. Biophys J. 1994 Aug;67(2):766–781. doi: 10.1016/S0006-3495(94)80537-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krauhs E., Little M., Kempf T., Hofer-Warbinek R., Ade W., Ponstingl H. Complete amino acid sequence of beta-tubulin from porcine brain. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4156–4160. doi: 10.1073/pnas.78.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kull F. J., Sablin E. P., Lau R., Fletterick R. J., Vale R. D. Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature. 1996 Apr 11;380(6574):550–555. doi: 10.1038/380550a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Q., Joshi H. C. gamma-tubulin is a minus end-specific microtubule binding protein. J Cell Biol. 1995 Oct;131(1):207–214. doi: 10.1083/jcb.131.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Little M., Krauhs E., Ponstingl H. Tubulin sequence conservation. Biosystems. 1981;14(3-4):239–246. doi: 10.1016/0303-2647(81)90031-9. [DOI] [PubMed] [Google Scholar]
  21. Lopez L. A., Sheetz M. P. Steric inhibition of cytoplasmic dynein and kinesin motility by MAP2. Cell Motil Cytoskeleton. 1993;24(1):1–16. doi: 10.1002/cm.970240102. [DOI] [PubMed] [Google Scholar]
  22. Marya P. K., Syed Z., Fraylich P. E., Eagles P. A. Kinesin and tau bind to distinct sites on microtubules. J Cell Sci. 1994 Jan;107(Pt 1):339–344. doi: 10.1242/jcs.107.1.339. [DOI] [PubMed] [Google Scholar]
  23. Mitchison T. J. Localization of an exchangeable GTP binding site at the plus end of microtubules. Science. 1993 Aug 20;261(5124):1044–1047. doi: 10.1126/science.8102497. [DOI] [PubMed] [Google Scholar]
  24. Nogales E., Wolf S. G., Downing K. H. Structure of the alpha beta tubulin dimer by electron crystallography. Nature. 1998 Jan 8;391(6663):199–203. doi: 10.1038/34465. [DOI] [PubMed] [Google Scholar]
  25. Paschal B. M., Obar R. A., Vallee R. B. Interaction of brain cytoplasmic dynein and MAP2 with a common sequence at the C terminus of tubulin. Nature. 1989 Nov 30;342(6249):569–572. doi: 10.1038/342569a0. [DOI] [PubMed] [Google Scholar]
  26. Ponstingl H., Krauhs E., Little M., Kempf T. Complete amino acid sequence of alpha-tubulin from porcine brain. Proc Natl Acad Sci U S A. 1981 May;78(5):2757–2761. doi: 10.1073/pnas.78.5.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rodionov V. I., Gyoeva F. K., Kashina A. S., Kuznetsov S. A., Gelfand V. I. Microtubule-associated proteins and microtubule-based translocators have different binding sites on tubulin molecule. J Biol Chem. 1990 Apr 5;265(10):5702–5707. [PubMed] [Google Scholar]
  28. Romberg L., Pierce D. W., Vale R. D. Role of the kinesin neck region in processive microtubule-based motility. J Cell Biol. 1998 Mar 23;140(6):1407–1416. doi: 10.1083/jcb.140.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roopnarine O., Thomas D. D. Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers. Biophys J. 1996 Jun;70(6):2795–2806. doi: 10.1016/S0006-3495(96)79849-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sablin E. P., Kull F. J., Cooke R., Vale R. D., Fletterick R. J. Crystal structure of the motor domain of the kinesin-related motor ncd. Nature. 1996 Apr 11;380(6574):555–559. doi: 10.1038/380555a0. [DOI] [PubMed] [Google Scholar]
  31. Sackett D. L., Bhattacharyya B., Wolff J. Tubulin subunit carboxyl termini determine polymerization efficiency. J Biol Chem. 1985 Jan 10;260(1):43–45. [PubMed] [Google Scholar]
  32. Sackett D. L., Wolff J. Proteolysis of tubulin and the substructure of the tubulin dimer. J Biol Chem. 1986 Jul 5;261(19):9070–9076. [PubMed] [Google Scholar]
  33. Schroer T. A., Steuer E. R., Sheetz M. P. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. doi: 10.1016/0092-8674(89)90627-2. [DOI] [PubMed] [Google Scholar]
  34. Serrano L., Avila J., Maccioni R. B. Controlled proteolysis of tubulin by subtilisin: localization of the site for MAP2 interaction. Biochemistry. 1984 Sep 25;23(20):4675–4681. doi: 10.1021/bi00315a024. [DOI] [PubMed] [Google Scholar]
  35. Serrano L., de la Torre J., Maccioni R. B., Avila J. Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5989–5993. doi: 10.1073/pnas.81.19.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  37. Svoboda K., Mitra P. P., Block S. M. Fluctuation analysis of motor protein movement and single enzyme kinetics. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11782–11786. doi: 10.1073/pnas.91.25.11782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Svoboda K., Schmidt C. F., Schnapp B. J., Block S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature. 1993 Oct 21;365(6448):721–727. doi: 10.1038/365721a0. [DOI] [PubMed] [Google Scholar]
  39. 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. 1997 Apr 4;272(14):8946–8956. doi: 10.1074/jbc.272.14.8946. [DOI] [PubMed] [Google Scholar]
  40. Tucker C., Goldstein L. S. Probing the kinesin-microtubule interaction. J Biol Chem. 1997 Apr 4;272(14):9481–9488. doi: 10.1074/jbc.272.14.9481. [DOI] [PubMed] [Google Scholar]
  41. Vale R. D., Funatsu T., Pierce D. W., Romberg L., Harada Y., Yanagida T. Direct observation of single kinesin molecules moving along microtubules. Nature. 1996 Apr 4;380(6573):451–453. doi: 10.1038/380451a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van Dijk J., Fernandez C., Chaussepied P. Effect of ATP analogues on the actin-myosin interface. Biochemistry. 1998 Jun 9;37(23):8385–8394. doi: 10.1021/bi980139a. [DOI] [PubMed] [Google Scholar]
  43. Wade R. H., Chrétien D. Cryoelectron microscopy of microtubules. J Struct Biol. 1993 Jan-Feb;110(1):1–27. doi: 10.1006/jsbi.1993.1001. [DOI] [PubMed] [Google Scholar]
  44. Wang Z., Khan S., Sheetz M. P. Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin. Biophys J. 1995 Nov;69(5):2011–2023. doi: 10.1016/S0006-3495(95)80071-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Z., Sheetz M. P. One-dimensional diffusion on microtubules of particles coated with cytoplasmic dynein and immunoglobulins. Cell Struct Funct. 1999 Oct;24(5):373–383. doi: 10.1247/csf.24.373. [DOI] [PubMed] [Google Scholar]
  46. White E. A., Burton P. R., Himes R. H. Polymorphic assembly of subtilisin-cleaved tubulin. Cell Motil Cytoskeleton. 1987;7(1):31–38. doi: 10.1002/cm.970070105. [DOI] [PubMed] [Google Scholar]
  47. Woehlke G., Ruby A. K., Hart C. L., Ly B., Hom-Booher N., Vale R. D. Microtubule interaction site of the kinesin motor. Cell. 1997 Jul 25;90(2):207–216. doi: 10.1016/s0092-8674(00)80329-3. [DOI] [PubMed] [Google Scholar]

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