Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1995 Jan;139(1):267–286. doi: 10.1093/genetics/139.1.267

Structural Analysis of Mutations in the Drosophila β2-Tubulin Isoform Reveals Regions in the β-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

J D Fackenthal 1, J A Hutchens 1, F R Turner 1, E C Raff 1
PMCID: PMC1206324  PMID: 7705629

Abstract

We have determined the lesions in a number of mutant alleles of βTub85D, the gene that encodes the testis-specific β2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the β2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all β-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate β-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that β3, a developmentally regulated Drosophila β-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type β2-tubulin. We show here by complementation analysis that β3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the β2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the β2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the β2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate β-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type β-tubulins. We propose that the integrity of this structure in the Drosophila testis β2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

Full Text

The Full Text of this article is available as a PDF (7.5 MB).

Selected References

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

  1. Barahona I., Soares H., Cyrne L., Penque D., Denoulet P., Rodrigues-Pousada C. Sequence of one alpha- and two beta-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other eukaryotic tubulin genes. J Mol Biol. 1988 Aug 5;202(3):365–382. doi: 10.1016/0022-2836(88)90271-9. [DOI] [PubMed] [Google Scholar]
  2. Bayley P. M., Schilstra M. J., Martin S. R. A simple formulation of microtubule dynamics: quantitative implications of the dynamic instability of microtubule populations in vivo and in vitro. J Cell Sci. 1989 Jun;93(Pt 2):241–254. doi: 10.1242/jcs.93.2.241. [DOI] [PubMed] [Google Scholar]
  3. Burns R. G., Surridge C. Analysis of beta-tubulin sequences reveals highly conserved, coordinated amino acid substitutions. Evidence that these 'hot spots' are directly involved in the conformational change required for dynamic instability. FEBS Lett. 1990 Oct 1;271(1-2):1–8. doi: 10.1016/0014-5793(90)80359-q. [DOI] [PubMed] [Google Scholar]
  4. Caplow M., Shanks J., Breidenbach S., Ruhlen R. L. Kinetics and mechanism of microtubule length changes by dynamic instability. J Biol Chem. 1988 Aug 5;263(22):10943–10951. [PubMed] [Google Scholar]
  5. Conner T. W., Thompson M. D., Silflow C. D. Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis. Gene. 1989 Dec 14;84(2):345–358. doi: 10.1016/0378-1119(89)90509-x. [DOI] [PubMed] [Google Scholar]
  6. Conzelmann K. K., Helftenbein E. Nucleotide sequence and expression of two beta-tubulin genes in Stylonychia lemnae. J Mol Biol. 1987 Dec 20;198(4):643–653. doi: 10.1016/0022-2836(87)90207-5. [DOI] [PubMed] [Google Scholar]
  7. Delves C. J., Ridley R. G., Goman M., Holloway S. P., Hyde J. E., Scaife J. G. Cloning of a beta-tubulin gene from Plasmodium falciparum. Mol Microbiol. 1989 Nov;3(11):1511–1519. doi: 10.1111/j.1365-2958.1989.tb00137.x. [DOI] [PubMed] [Google Scholar]
  8. Derbyshire V., Grindley N. D., Joyce C. M. The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. EMBO J. 1991 Jan;10(1):17–24. doi: 10.1002/j.1460-2075.1991.tb07916.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Di Bernardo M. G., Gianguzza F., Ciaccio M., Palla F., Colombo P., Di Blasi F., Fais M., Spinelli G. Nucleotide sequence of a full length cDNA clone encoding for beta-tubulin of the sea urchin Paracentrotus lividus. Nucleic Acids Res. 1989 Jul 25;17(14):5851–5851. doi: 10.1093/nar/17.14.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fuller M. T., Caulton J. H., Hutchens J. A., Kaufman T. C., Raff E. C. Genetic analysis of microtubule structure: a beta-tubulin mutation causes the formation of aberrant microtubules in vivo and in vitro. J Cell Biol. 1987 Mar;104(3):385–394. doi: 10.1083/jcb.104.3.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuller M. T., Caulton J. H., Hutchens J. A., Kaufman T. C., Raff E. C. Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays. J Cell Biol. 1988 Jul;107(1):141–152. doi: 10.1083/jcb.107.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gay D. A., Yen T. J., Lau J. T., Cleveland D. W. Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA. Cell. 1987 Aug 28;50(5):671–679. doi: 10.1016/0092-8674(87)90325-4. [DOI] [PubMed] [Google Scholar]
  13. Ginzburg I., Teichman A., Dodemont H. J., Behar L., Littauer U. Z. Regulation of three beta-tubulin mRNAs during rat brain development. EMBO J. 1985 Dec 30;4(13B):3667–3673. doi: 10.1002/j.1460-2075.1985.tb04133.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Good P. J., Richter K., Dawid I. B. The sequence of a nervous system-specific, class II beta-tubulin gene from Xenopus laevis. Nucleic Acids Res. 1989 Oct 11;17(19):8000–8000. doi: 10.1093/nar/17.19.8000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gu W., Cowan N. J. Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains. Mol Cell Biol. 1989 Aug;9(8):3418–3428. doi: 10.1128/mcb.9.8.3418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hall J. L., Dudley L., Dobner P. R., Lewis S. A., Cowan N. J. Identification of two human beta-tubulin isotypes. Mol Cell Biol. 1983 May;3(5):854–862. doi: 10.1128/mcb.3.5.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harlow P., Litwin S., Nemer M. Synonymous nucleotide substitution rates of beta-tubulin and histone genes conform to high overall genomic rates in rodents but not in sea urchins. J Mol Evol. 1988;27(1):56–64. doi: 10.1007/BF02099730. [DOI] [PubMed] [Google Scholar]
  18. Harper J. F., Mages W. Organization and structure of Volvox beta-tubulin genes. Mol Gen Genet. 1988 Aug;213(2-3):315–324. doi: 10.1007/BF00339597. [DOI] [PubMed] [Google Scholar]
  19. Harris G. S., Keath E. J., Medoff J. Characterization of alpha and beta tubulin genes in the dimorphic fungus Histoplasma capsulatum. J Gen Microbiol. 1989 Jul;135(7):1817–1832. doi: 10.1099/00221287-135-7-1817. [DOI] [PubMed] [Google Scholar]
  20. Hays T. S., Deuring R., Robertson B., Prout M., Fuller M. T. Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster. Mol Cell Biol. 1989 Mar;9(3):875–884. doi: 10.1128/mcb.9.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hiraoka Y., Toda T., Yanagida M. The NDA3 gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell. 1984 Dec;39(2 Pt 1):349–358. doi: 10.1016/0092-8674(84)90013-8. [DOI] [PubMed] [Google Scholar]
  22. Hoyle H. D., Raff E. C. Two Drosophila beta tubulin isoforms are not functionally equivalent. J Cell Biol. 1990 Sep;111(3):1009–1026. doi: 10.1083/jcb.111.3.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kanaya S., Kohara A., Miura Y., Sekiguchi A., Iwai S., Inoue H., Ohtsuka E., Ikehara M. Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis. J Biol Chem. 1990 Mar 15;265(8):4615–4621. [PubMed] [Google Scholar]
  24. Kemphues K. J., Kaufman T. C., Raff R. A., Raff E. C. The testis-specific beta-tubulin subunit in Drosophila melanogaster has multiple functions in spermatogenesis. Cell. 1982 Dec;31(3 Pt 2):655–670. doi: 10.1016/0092-8674(82)90321-x. [DOI] [PubMed] [Google Scholar]
  25. Kemphues K. J., Raff E. C., Kaufman T. C. Genetic analysis of B2t, the structural gene for a testis-specific beta-tubulin subunit in Drosophila melanogaster. Genetics. 1983 Oct;105(2):345–356. doi: 10.1093/genetics/105.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kemphues K. J., Raff R. A., Kaufman T. C., Raff E. C. Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3991–3995. doi: 10.1073/pnas.76.8.3991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kimble M., Dettman R. W., Raff E. C. The beta 3-tubulin gene of Drosophila melanogaster is essential for viability and fertility. Genetics. 1990 Dec;126(4):991–1005. doi: 10.1093/genetics/126.4.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kimble M., Incardona J. P., Raff E. C. A variant beta-tubulin isoform of Drosophila melanogaster (beta 3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules. Dev Biol. 1989 Feb;131(2):415–429. doi: 10.1016/s0012-1606(89)80014-4. [DOI] [PubMed] [Google Scholar]
  29. Kimmel B. E., Samson S., Wu J., Hirschberg R., Yarbrough L. R. Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense:nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene. 1985;35(3):237–248. doi: 10.1016/0378-1119(85)90002-2. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee M. G., Lewis S. A., Wilde C. D., Cowan N. J. Evolutionary history of a multigene family: an expressed human beta-tubulin gene and three processed pseudogenes. Cell. 1983 Jun;33(2):477–487. doi: 10.1016/0092-8674(83)90429-4. [DOI] [PubMed] [Google Scholar]
  33. Lee M. G., Loomis C., Cowan N. J. Sequence of an expressed human beta-tubulin gene containing ten Alu family members. Nucleic Acids Res. 1984 Jul 25;12(14):5823–5836. doi: 10.1093/nar/12.14.5823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lewis S. A., Lee M. G., Cowan N. J. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol. 1985 Sep;101(3):852–861. doi: 10.1083/jcb.101.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mandelkow E. M., Mandelkow E. Microtubule oscillations. Cell Motil Cytoskeleton. 1992;22(4):235–244. doi: 10.1002/cm.970220403. [DOI] [PubMed] [Google Scholar]
  36. Matthews K. A., Kaufman T. C. Developmental consequences of mutations in the 84B alpha-tubulin gene of Drosophila melanogaster. Dev Biol. 1987 Jan;119(1):100–114. doi: 10.1016/0012-1606(87)90211-9. [DOI] [PubMed] [Google Scholar]
  37. May G. S., Tsang M. L., Smith H., Fidel S., Morris N. R. Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene. 1987;55(2-3):231–243. doi: 10.1016/0378-1119(87)90283-6. [DOI] [PubMed] [Google Scholar]
  38. Michiels F., Falkenburg D., Müller A. M., Hinz U., Otto U., Bellmann R., Glätzer K. H., Brand R., Bialojan S., Renkawitz-Pohl R. Testis-specific beta 2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous beta 1 tubulin. Chromosoma. 1987;95(6):387–395. doi: 10.1007/BF00333989. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Monteiro M. J., Cleveland D. W. Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes. J Mol Biol. 1988 Feb 5;199(3):439–446. doi: 10.1016/0022-2836(88)90616-x. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Oppenheimer D. G., Haas N., Silflow C. D., Snustad D. P. The beta-tubulin gene family of Arabidopsis thaliana: preferential accumulation of the beta 1 transcript in roots. Gene. 1988;63(1):87–102. doi: 10.1016/0378-1119(88)90548-3. [DOI] [PubMed] [Google Scholar]
  43. Orbach M. J., Porro E. B., Yanofsky C. Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol. 1986 Jul;6(7):2452–2461. doi: 10.1128/mcb.6.7.2452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Praitis V., Katz W. S., Solomon F. A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Sep;11(9):4726–4731. doi: 10.1128/mcb.11.9.4726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rudolph J. E., Kimble M., Hoyle H. D., Subler M. A., Raff E. C. Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function. Mol Cell Biol. 1987 Jun;7(6):2231–2242. doi: 10.1128/mcb.7.6.2231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Savage C., Hamelin M., Culotti J. G., Coulson A., Albertson D. G., Chalfie M. mec-7 is a beta-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans. Genes Dev. 1989 Jun;3(6):870–881. doi: 10.1101/gad.3.6.870. [DOI] [PubMed] [Google Scholar]
  47. Schantz M. L., Schantz R. Sequence of a cDNA clone encoding beta tubulin from Euglena gracilis. Nucleic Acids Res. 1989 Aug 25;17(16):6727–6727. doi: 10.1093/nar/17.16.6727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Singhofer-Wowra M., Clayton L., Dawson P., Gull K., Little M. Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae. Eur J Biochem. 1986 Dec 15;161(3):669–679. doi: 10.1111/j.1432-1033.1986.tb10492.x. [DOI] [PubMed] [Google Scholar]
  49. Smith H. A., Allaudeen H. S., Whitman M. H., Koltin Y., Gorman J. A. Isolation and characterization of a beta-tubulin gene from Candida albicans. Gene. 1988;63(1):53–63. doi: 10.1016/0378-1119(88)90545-8. [DOI] [PubMed] [Google Scholar]
  50. Stewart R. J., Farrell K. W., Wilson L. Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism. Biochemistry. 1990 Jul 10;29(27):6489–6498. doi: 10.1021/bi00479a022. [DOI] [PubMed] [Google Scholar]
  51. Sánchez F., Natzle J. E., Cleveland D. W., Kirschner M. W., McCarthy B. J. A dispersed multigene family encoding tubulin in Drosophila melanogaster. Cell. 1980 Dec;22(3):845–854. doi: 10.1016/0092-8674(80)90561-9. [DOI] [PubMed] [Google Scholar]
  52. Yen T. J., Gay D. A., Pachter J. S., Cleveland D. W. Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides. Mol Cell Biol. 1988 Mar;8(3):1224–1235. doi: 10.1128/mcb.8.3.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yen T. J., Machlin P. S., Cleveland D. W. Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin. Nature. 1988 Aug 18;334(6183):580–585. doi: 10.1038/334580a0. [DOI] [PubMed] [Google Scholar]
  54. Youngblom J., Schloss J. A., Silflow C. D. The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins. Mol Cell Biol. 1984 Dec;4(12):2686–2696. doi: 10.1128/mcb.4.12.2686. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES