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. 1987 Oct;7(10):3792–3798. doi: 10.1128/mcb.7.10.3792

Domains of beta-tubulin essential for conserved functions in vivo.

J L Fridovich-Keil 1, J F Bond 1, F Solomon 1
PMCID: PMC368036  PMID: 2891028

Abstract

The relationship between the primary sequence of tubulins and their properties in cells was studied by gene transfection experiments. Previously, we studied a chimeric beta-tubulin formed from chicken beta-tubulin-2 sequences in the amino-terminal portion and the highly divergent Saccharomyces cerevisiae TUB2 sequences in the carboxy-terminal 25% of the molecule. In the cytoplasm of cultured animal cells, this protein incorporates into all microtubule structures and assembles with the same efficiency as endogenous tubulin. We show that the protein products of chimeric genes with an increasing proportion of yeast sequence, extending 5' of the carboxy-terminal 25%, are abnormal in two ways. First, they assemble with a significantly lower efficiency than the original chimeric protein or the endogenous tubulins. Second, they are less stable in the cytoplasm. The results suggest that the position of the yeast sequences is crucial in determining the properties of the molecule. Results of analyses of 1 deletion mutation and 10 linker insertions in the original chimeric tubulin suggest that those changes made outside the carboxyl terminus completely disrupt assembly activity, while those made in the carboxyl terminus do not.

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Selected References

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

  1. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  2. Breitling F., Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986 May 20;189(2):367–370. doi: 10.1016/0022-2836(86)90517-6. [DOI] [PubMed] [Google Scholar]
  3. Brown H. R., Erickson H. P. Assembly of proteolytically cleaved tubulin. Arch Biochem Biophys. 1983 Jan;220(1):46–51. doi: 10.1016/0003-9861(83)90385-5. [DOI] [PubMed] [Google Scholar]
  4. Cleveland D. W., Sullivan K. F. Molecular biology and genetics of tubulin. Annu Rev Biochem. 1985;54:331–365. doi: 10.1146/annurev.bi.54.070185.001555. [DOI] [PubMed] [Google Scholar]
  5. Davies J., Jimenez A. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1089–1092. doi: 10.4269/ajtmh.1980.29.1089. [DOI] [PubMed] [Google Scholar]
  6. Fine R. E., Taylor L. Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus. Exp Cell Res. 1976 Oct 1;102(1):162–168. doi: 10.1016/0014-4827(76)90311-6. [DOI] [PubMed] [Google Scholar]
  7. Grollman A. P. Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1867–1874. doi: 10.1073/pnas.56.6.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guo L. H., Wu R. New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis. Nucleic Acids Res. 1982 Mar 25;10(6):2065–2084. doi: 10.1093/nar/10.6.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kim S., Magendantz M., Katz W., Solomon F. Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo. J Cell Biol. 1987 Jan;104(1):51–59. doi: 10.1083/jcb.104.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kirchner K., Mandelkow E. M. Tubulin domains responsible for assembly of dimers and protofilaments. EMBO J. 1985 Sep;4(9):2397–2402. doi: 10.1002/j.1460-2075.1985.tb03945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lewis S. A., Gu W., Cowan N. J. Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules. Cell. 1987 May 22;49(4):539–548. doi: 10.1016/0092-8674(87)90456-9. [DOI] [PubMed] [Google Scholar]
  12. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ludueńa R. F., Shooter E. M., Wilson L. Structure of the tubulin dimer. J Biol Chem. 1977 Oct 25;252(20):7006–7014. [PubMed] [Google Scholar]
  14. Maccioni R. B., Serrano L., Avila J., Cann J. R. Characterization and structural aspects of the enhanced assembly of tubulin after removal of its carboxyl-terminal domain. Eur J Biochem. 1986 Apr 15;156(2):375–381. doi: 10.1111/j.1432-1033.1986.tb09593.x. [DOI] [PubMed] [Google Scholar]
  15. Mandelkow E. M., Herrmann M., Rühl U. Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J Mol Biol. 1985 Sep 20;185(2):311–327. doi: 10.1016/0022-2836(85)90406-1. [DOI] [PubMed] [Google Scholar]
  16. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  18. Ng M., Privalsky M. L. Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis. J Virol. 1986 May;58(2):542–553. doi: 10.1128/jvi.58.2.542-553.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Schatz P. J., Georges G. E., Solomon F., Botstein D. Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo. Mol Cell Biol. 1987 Oct;7(10):3799–3805. doi: 10.1128/mcb.7.10.3799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. doi: 10.1038/277665a0. [DOI] [PubMed] [Google Scholar]
  24. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  25. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  26. Theurkauf W. E., Baum H., Bo J., Wensink P. C. Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8477–8481. doi: 10.1073/pnas.83.22.8477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Valenzuela P., Quiroga M., Zaldivar J., Rutter W. J., Kirschner M. W., Cleveland D. W. Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs. Nature. 1981 Feb 19;289(5799):650–655. doi: 10.1038/289650a0. [DOI] [PubMed] [Google Scholar]
  29. Zieve G., Solomon F. Proteins specifically associated with the microtubules of the mammalian mitotic spindle. Cell. 1982 Feb;28(2):233–242. doi: 10.1016/0092-8674(82)90341-5. [DOI] [PubMed] [Google Scholar]

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