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. 1990 Jul;87(13):5041–5045. doi: 10.1073/pnas.87.13.5041

Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein.

G W Farr 1, M B Yaffe 1, H Sternlicht 1
PMCID: PMC54257  PMID: 2367522

Abstract

Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Caplow M., Brylawski B. P., Reid R. Mechanism for nucleotide incorporation into steady-state microtubules. Biochemistry. 1984 Dec 18;23(26):6745–6752. doi: 10.1021/bi00321a071. [DOI] [PubMed] [Google Scholar]
  2. Correia J. J., Baty L. T., Williams R. C., Jr Mg2+ dependence of guanine nucleotide binding to tubulin. J Biol Chem. 1987 Dec 25;262(36):17278–17284. [PubMed] [Google Scholar]
  3. Correia J. J., Beth A. H., Williams R. C., Jr Tubulin exchanges divalent cations at both guanine nucleotide-binding sites. J Biol Chem. 1988 Aug 5;263(22):10681–10686. [PubMed] [Google Scholar]
  4. Detrich H. W., 3rd, Williams R. C. Reversible dissociation of the alpha beta dimer of tubulin from bovine brain. Biochemistry. 1978 Sep 19;17(19):3900–3907. doi: 10.1021/bi00612a002. [DOI] [PubMed] [Google Scholar]
  5. Duanmu C., Lin C. M., Hamel E. Tubulin polymerization with ATP is mediated through the exchangeable GTP site. Biochim Biophys Acta. 1986 Mar 19;881(1):113–123. doi: 10.1016/0304-4165(86)90104-2. [DOI] [PubMed] [Google Scholar]
  6. Fishback J. L., Yarbrough L. R. Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects. J Biol Chem. 1984 Feb 10;259(3):1968–1973. [PubMed] [Google Scholar]
  7. Geahlen R. L., Haley B. E. Use of a GTP photoaffinity probe to resolve aspects of the mechanism of tubulin polymerization. J Biol Chem. 1979 Dec 10;254(23):11982–11987. [PubMed] [Google Scholar]
  8. Higashijima T., Ferguson K. M., Sternweis P. C., Smigel M. D., Gilman A. G. Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins. J Biol Chem. 1987 Jan 15;262(2):762–766. [PubMed] [Google Scholar]
  9. Huang A. B., Lin C. M., Hamel E. Differential effects of magnesium on tubulin-nucleotide interactions. Biochim Biophys Acta. 1985 Nov 8;832(1):22–32. doi: 10.1016/0167-4838(85)90170-0. [DOI] [PubMed] [Google Scholar]
  10. Jemiolo D. K., Grisham C. M. Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin. J Biol Chem. 1982 Jul 25;257(14):8148–8152. [PubMed] [Google Scholar]
  11. Manser E. J., Bayley P. M. Conformational and assembly properties of nucleotide-depleted tubulin. Biophys J. 1986 Jan;49(1):81–83. doi: 10.1016/S0006-3495(86)83602-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Manser E. J., Bayley P. M. Tubulin-nucleotide interactions. Effects of removal of exchangeable guanine nucleotide on protein conformation and microtubule assembly. Biochem J. 1987 Jan 1;241(1):105–110. doi: 10.1042/bj2410105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Margolis R. L., Wilson L. Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell. 1978 Jan;13(1):1–8. doi: 10.1016/0092-8674(78)90132-0. [DOI] [PubMed] [Google Scholar]
  14. Mitchison T. J. Microtubule dynamics and kinetochore function in mitosis. Annu Rev Cell Biol. 1988;4:527–549. doi: 10.1146/annurev.cb.04.110188.002523. [DOI] [PubMed] [Google Scholar]
  15. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  16. Nath J. P., Eagle G. R., Himes R. H. Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate. Biochemistry. 1985 Mar 12;24(6):1555–1560. doi: 10.1021/bi00327a040. [DOI] [PubMed] [Google Scholar]
  17. Sackett D. L., Zimmerman D. A., Wolff J. Tubulin dimer dissociation and proteolytic accessibility. Biochemistry. 1989 Mar 21;28(6):2662–2667. doi: 10.1021/bi00432a045. [DOI] [PubMed] [Google Scholar]
  18. Terry B. J., Purich D. L. Nucleotide-dependent enzymes associated with microtubule systems. Adv Enzymol Relat Areas Mol Biol. 1982;53:113–161. doi: 10.1002/9780470122983.ch4. [DOI] [PubMed] [Google Scholar]
  19. Walker R. A., O'Brien E. T., Pryer N. K., Soboeiro M. F., Voter W. A., Erickson H. P., Salmon E. D. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J Cell Biol. 1988 Oct;107(4):1437–1448. doi: 10.1083/jcb.107.4.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
  21. Yaffe M. B., Farr G. W., Sternlicht H. Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers. J Biol Chem. 1989 Nov 15;264(32):19045–19051. [PubMed] [Google Scholar]
  22. Yaffe M. B., Farr G. W., Sternlicht H. Translation of beta-tubulin mRNA in vitro generates multiple molecular forms. J Biol Chem. 1988 Nov 5;263(31):16023–16031. [PubMed] [Google Scholar]
  23. Zabrecky J. R., Cole R. D. Binding of ATP to tubulin. Nature. 1982 Apr 22;296(5859):775–776. doi: 10.1038/296775a0. [DOI] [PubMed] [Google Scholar]
  24. Zabrecky J. R., Cole R. D. Localization of the ATP binding site on alpha-tubulin. Arch Biochem Biophys. 1983 Sep;225(2):475–481. doi: 10.1016/0003-9861(83)90056-5. [DOI] [PubMed] [Google Scholar]
  25. Zeeberg B., Caplow M. Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions. Biochemistry. 1979 Sep 4;18(18):3880–3886. doi: 10.1021/bi00585a007. [DOI] [PubMed] [Google Scholar]

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