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. 1988 Jul 1;107(1):121–131. doi: 10.1083/jcb.107.1.121

Purification and characterization of a basal body-associated Ca2+- binding protein

PMCID: PMC2115177  PMID: 3292538

Abstract

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.

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Selected References

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  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amos W. B. Contraction and calcium binding in the vorticellid ciliates. Soc Gen Physiol Ser. 1975;30:411–436. [PubMed] [Google Scholar]
  3. Bidlingmeyer B. A., Cohen S. A., Tarvin T. L. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr. 1984 Dec 7;336(1):93–104. doi: 10.1016/s0378-4347(00)85133-6. [DOI] [PubMed] [Google Scholar]
  4. Bolduc C., Lee V. D., Huang B. Beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1988 Jan;85(1):131–135. doi: 10.1073/pnas.85.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brenner S. L., Brinkley B. R. Tubulin assembly sites and the organization of microtubule arrays in mammalian cells. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):241–254. doi: 10.1101/sqb.1982.046.01.027. [DOI] [PubMed] [Google Scholar]
  6. Brinkley B. R. Microtubule organizing centers. Annu Rev Cell Biol. 1985;1:145–172. doi: 10.1146/annurev.cb.01.110185.001045. [DOI] [PubMed] [Google Scholar]
  7. Burgess W. H. Characterization of calmodulin and calmodulin isotypes from sea urchin gametes. J Biol Chem. 1982 Feb 25;257(4):1800–1804. [PubMed] [Google Scholar]
  8. Charbonneau H., Cormier M. J. Purification of plant calmodulin by fluphenazine-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1979 Oct 12;90(3):1039–1047. doi: 10.1016/0006-291x(79)91931-4. [DOI] [PubMed] [Google Scholar]
  9. Coss R. A. Mitosis in Chlamydomonas reinhardtii basal bodies and the mitotic apparatus. J Cell Biol. 1974 Oct;63(1):325–329. doi: 10.1083/jcb.63.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gitelman S. E., Witman G. B. Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella. J Cell Biol. 1980 Dec;87(3 Pt 1):764–770. doi: 10.1083/jcb.87.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  12. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gould R. R. The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization. J Cell Biol. 1975 Apr;65(1):65–74. doi: 10.1083/jcb.65.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heidemann S. R., Sander G., Kirschner M. W. Evidence for a functional role of RNA in centrioles. Cell. 1977 Mar;10(3):337–350. doi: 10.1016/0092-8674(77)90021-6. [DOI] [PubMed] [Google Scholar]
  15. Hoops H. J., Witman G. B. Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella. J Cell Biol. 1983 Sep;97(3):902–908. doi: 10.1083/jcb.97.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang B., Mengersen A., Lee V. D. Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product. J Cell Biol. 1988 Jul;107(1):133–140. doi: 10.1083/jcb.107.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  18. Jamieson G. A., Jr, Vanaman T. C. Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine. Biochem Biophys Res Commun. 1979 Oct 12;90(3):1048–1056. doi: 10.1016/0006-291x(79)91932-6. [DOI] [PubMed] [Google Scholar]
  19. Johnson U. G., Porter K. R. Fine structure of cell division in Chlamydomonas reinhardi. Basal bodies and microtubules. J Cell Biol. 1968 Aug;38(2):403–425. doi: 10.1083/jcb.38.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  21. Kretsinger R. H. Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980;8(2):119–174. doi: 10.3109/10409238009105467. [DOI] [PubMed] [Google Scholar]
  22. Kuroiwa T., Kawano S., Nishibayashi S., Sato C. Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature. 1982 Jul 29;298(5873):481–483. doi: 10.1038/298481a0. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lewin R. A., Lounsbery D. M. Isolation, cultivation and characterization of flexibacteria. J Gen Microbiol. 1969 Oct;58(2):145–170. doi: 10.1099/00221287-58-2-145. [DOI] [PubMed] [Google Scholar]
  25. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  26. Lukas T. J., Wiggins M. E., Watterson D. M. Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas. Plant Physiol. 1985 Jul;78(3):477–483. doi: 10.1104/pp.78.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marshak D. R., Clarke M., Roberts D. M., Watterson D. M. Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum. Biochemistry. 1984 Jun 19;23(13):2891–2899. doi: 10.1021/bi00308a007. [DOI] [PubMed] [Google Scholar]
  28. Mazia D. The chromosome cycle and the centrosome cycle in the mitotic cycle. Int Rev Cytol. 1987;100:49–92. doi: 10.1016/s0074-7696(08)61698-8. [DOI] [PubMed] [Google Scholar]
  29. McFadden G. I., Schulze D., Surek B., Salisbury J. L., Melkonian M. Basal body reorientation mediated by a Ca2+-modulated contractile protein. J Cell Biol. 1987 Aug;105(2):903–912. doi: 10.1083/jcb.105.2.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Melkonian M. Ultrastructural aspects of basal body associated fibrous structures in green algae: a critical review. Biosystems. 1980;12(1-2):85–104. doi: 10.1016/0303-2647(80)90040-4. [DOI] [PubMed] [Google Scholar]
  31. Piperno G., Luck D. J. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J Biol Chem. 1979 Apr 25;254(8):3084–3090. [PubMed] [Google Scholar]
  32. Piperno G., Luck D. J. Inner arm dyneins from flagella of Chlamydomonas reinhardtii. Cell. 1981 Dec;27(2 Pt 1):331–340. doi: 10.1016/0092-8674(81)90416-5. [DOI] [PubMed] [Google Scholar]
  33. Ringo D. L. Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J Cell Biol. 1967 Jun;33(3):543–571. doi: 10.1083/jcb.33.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Salisbury J. L., Baron A. T., Coling D. E., Martindale V. E., Sanders M. A. Calcium-modulated contractile proteins associated with the eucaryotic centrosome. Cell Motil Cytoskeleton. 1986;6(2):193–197. doi: 10.1002/cm.970060218. [DOI] [PubMed] [Google Scholar]
  35. Salisbury J. L., Baron A., Surek B., Melkonian M. Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle. J Cell Biol. 1984 Sep;99(3):962–970. doi: 10.1083/jcb.99.3.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Salisbury J. L., Sanders M. A., Harpst L. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii. J Cell Biol. 1987 Oct;105(4):1799–1805. doi: 10.1083/jcb.105.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schaefer W. H., Lukas T. J., Blair I. A., Schultz J. E., Watterson D. M. Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain. J Biol Chem. 1987 Jan 25;262(3):1025–1029. [PubMed] [Google Scholar]
  38. Segal R. A., Huang B., Ramanis Z., Luck D. J. Mutant strains of Chlamydomonas reinhardtii that move backwards only. J Cell Biol. 1984 Jun;98(6):2026–2034. doi: 10.1083/jcb.98.6.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Snell W. J., Dentler W. L., Haimo L. T., Binder L. I., Rosenbaum J. L. Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi. Science. 1974 Jul 26;185(4148):357–360. doi: 10.1126/science.185.4148.357. [DOI] [PubMed] [Google Scholar]
  40. Snell W. J. Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes. Exp Cell Res. 1982 Mar;138(1):109–119. doi: 10.1016/0014-4827(82)90096-9. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Van Eldik L. J., Piperno G., Watterson D. M. Similarities and dissimilarities between calmodulin and a Chlamydomonas flagellar protein. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4779–4783. doi: 10.1073/pnas.77.8.4779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Van Eldik L. J., Watterson D. M. Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site. J Biol Chem. 1981 May 10;256(9):4205–4210. [PubMed] [Google Scholar]
  44. Van Eldik L. J., Zendegui J. G., Marshak D. R., Watterson D. M. Calcium-binding proteins and the molecular basis of calcium action. Int Rev Cytol. 1982;77:1–61. doi: 10.1016/s0074-7696(08)62463-8. [DOI] [PubMed] [Google Scholar]
  45. Weiss B., Levin R. M. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303. [PubMed] [Google Scholar]
  46. Weiss R. L. Ultrastructure of the flagellar roots in Chlamydomonas gametes. J Cell Sci. 1984 Apr;67:133–143. doi: 10.1242/jcs.67.1.133. [DOI] [PubMed] [Google Scholar]
  47. Wright R. L., Salisbury J., Jarvik J. W. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation. J Cell Biol. 1985 Nov;101(5 Pt 1):1903–1912. doi: 10.1083/jcb.101.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]

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