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. 1986 May 1;102(5):1671–1678. doi: 10.1083/jcb.102.5.1671

Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation

PMCID: PMC2114234  PMID: 3700472

Abstract

Flagellates of Naegleria gruberi contain two calmodulins that differ in apparent molecular weight and intracellular location. Calmodulin-1, localized in flagella, has an apparent molecular weight of approximately 16,000, approximately the size of other protozoan calmodulins, whereas calmodulin-2, localized in cell bodies, is 15,300. Both proteins, purified, are calmodulins by several criteria, including Ca2+-dependent stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase and affinity for antibodies to vertebrate calmodulin. The finding of two calmodulins is unusual. Since the only known difference is apparent molecular weight, one calmodulin could be derived from the other, except that both calmodulins are synthesized in a wheat germ, cell-free system directed by RNA from differentiating Naegleria. Translatable mRNAs encoding calmodulins 1 and 2, not detected in amebas, appear and subsequently disappear concurrently during the 100-min differentiation of Naegleria from amebas to flagellates. Furthermore, these mRNAs increase and then decrease in abundance concurrently with those for flagellar tubulins, which suggests the possibility that the expression of the unrelated genes for calmodulin and tubulin may be under coordinate control during differentiation.

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Selected References

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  1. Bazari W. L., Clarke M. Characterization of a novel calmodulin from Dictyostelium discoideum. J Biol Chem. 1981 Apr 10;256(7):3598–3603. [PubMed] [Google Scholar]
  2. 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]
  3. Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
  4. Chafouleas J. G., Dedman J. R., Munjaal R. P., Means A. R. Calmodulin. Development and application of a sensitive radioimmunoassay. J Biol Chem. 1979 Oct 25;254(20):10262–10267. [PubMed] [Google Scholar]
  5. Chafouleas J. G., Lagacé L., Bolton W. E., Boyd A. E., 3rd, Means A. R. Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle. Cell. 1984 Jan;36(1):73–81. doi: 10.1016/0092-8674(84)90075-8. [DOI] [PubMed] [Google Scholar]
  6. Chafouleas J. G., Pardue R. L., Brinkley B. R., Dedman J. R., Means A. R. Regulation of intracellular levels of calmodulin and tubulin in normal and transformed cells. Proc Natl Acad Sci U S A. 1981 Feb;78(2):996–1000. doi: 10.1073/pnas.78.2.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chafouleas J. G., Riser M. E., Lagacé L., Means A. R. Production of polyclonal and monoclonal antibodies to calmodulin and utilization of these immunological probes. Methods Enzymol. 1983;102:104–110. doi: 10.1016/s0076-6879(83)02011-x. [DOI] [PubMed] [Google Scholar]
  8. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  9. Chien Y. H., Dawid I. B. Isolation and characterization of calmodulin genes from Xenopus laevis. Mol Cell Biol. 1984 Mar;4(3):507–513. doi: 10.1128/mcb.4.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke M., Bazari W. L., Kayman S. C. Isolation and properties of calmodulin from Dictyostelium discoideum. J Bacteriol. 1980 Jan;141(1):397–400. doi: 10.1128/jb.141.1.397-400.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eldik L. J., Grossman A. R., Iverson D. B., Watterson D. M. Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1912–1916. doi: 10.1073/pnas.77.4.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fulton C. Cell differentiation in Naegleria gruberi. Annu Rev Microbiol. 1977;31:597–629. doi: 10.1146/annurev.mi.31.100177.003121. [DOI] [PubMed] [Google Scholar]
  13. Fulton C. Intracellular regulation of cell shape and motility in Naegleria. First insights and a working hypothesis. J Supramol Struct. 1977;6(1):13–43. doi: 10.1002/jss.400060103. [DOI] [PubMed] [Google Scholar]
  14. Fulton C., Walsh C. Cell differentiation and flagellar elongation in Naegleria gruberi. Dependence on transcription and translation. J Cell Biol. 1980 May;85(2):346–360. doi: 10.1083/jcb.85.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Grab D. J., Berzins K., Cohen R. S., Siekevitz P. Presence of calmodulin in postsynaptic densities isolated from canine cerebral cortex. J Biol Chem. 1979 Sep 10;254(17):8690–8696. [PubMed] [Google Scholar]
  18. Greaser M. L., Gergely J. Purification and properties of the components from troponin. J Biol Chem. 1973 Mar 25;248(6):2125–2133. [PubMed] [Google Scholar]
  19. Jamieson G. A., Jr, Frazier W. A. Dictyostelium calmodulin: affinity isolation and characterization. Arch Biochem Biophys. 1983 Dec;227(2):609–617. doi: 10.1016/0003-9861(83)90490-3. [DOI] [PubMed] [Google Scholar]
  20. Jamieson G. A., Jr, Vanaman T. C., Blum J. J. Presence of calmodulin in Tetrahymena. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6471–6475. doi: 10.1073/pnas.76.12.6471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kakiuchi S., Sobue K., Yamazaki R., Nagao S., Umeki S., Nozawa Y., Yazawa M., Yagi K. Ca2+-dependent modulator proteins from Tetrahymena pyriformis, sea anemone, and scallop and guanylate cyclase activation. J Biol Chem. 1981 Jan 10;256(1):19–22. [PubMed] [Google Scholar]
  22. Klee C. B., Vanaman T. C. Calmodulin. Adv Protein Chem. 1982;35:213–321. doi: 10.1016/s0065-3233(08)60470-2. [DOI] [PubMed] [Google Scholar]
  23. Kowit J. D., Fulton C. Programmed synthesis of tubulin for the flagella that develop during cell differentiation in Naegleria gruberi. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2877–2881. doi: 10.1073/pnas.71.7.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kowit J. D., Fulton C. Purification and properties of flagellar outer doublet tubulin from Naegleria gruberi and a radioimmune assay for tubulin. J Biol Chem. 1974 Jun 10;249(11):3638–3646. [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. 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]
  27. Lai E. Y., Walsh C., Wardell D., Fulton C. Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria. Cell. 1979 Aug;17(4):867–878. doi: 10.1016/0092-8674(79)90327-1. [DOI] [PubMed] [Google Scholar]
  28. Manalan A. S., Klee C. B. Calmodulin. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;18:227–278. [PubMed] [Google Scholar]
  29. 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]
  30. Nagao S., Banno Y., Nozawa Y., Sobue K., Yamazaki R., Kakiuchi S. Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis. J Biochem. 1981 Sep;90(3):897–899. doi: 10.1093/oxfordjournals.jbchem.a133549. [DOI] [PubMed] [Google Scholar]
  31. Ohnishi K., Suzuki Y., Watanabe Y. Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium. Exp Cell Res. 1982 Jan;137(1):217–227. doi: 10.1016/0014-4827(82)90022-2. [DOI] [PubMed] [Google Scholar]
  32. Putkey J. A., Slaughter G. R., Means A. R. Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. J Biol Chem. 1985 Apr 25;260(8):4704–4712. [PubMed] [Google Scholar]
  33. Putkey J. A., Ts'ui K. F., Tanaka T., Lagacé L., Stein J. P., Lai E. C., Means A. R. Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone. J Biol Chem. 1983 Oct 10;258(19):11864–11870. [PubMed] [Google Scholar]
  34. Schleicher M., Lukas T. J., Watterson D. M. Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii. Arch Biochem Biophys. 1984 Feb 15;229(1):33–42. doi: 10.1016/0003-9861(84)90127-9. [DOI] [PubMed] [Google Scholar]
  35. Sussman D. J., Lai E. Y., Fulton C. Rapid disappearance of translatable actin mRNA during cell differentiation in Naegleria. J Biol Chem. 1984 Jun 10;259(11):7355–7360. [PubMed] [Google Scholar]
  36. 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]
  37. Wallace R. W., Tallant E. A., Cheung W. Y. Assay of calmodulin by Ca2+-dependent phosphodiesterase. Methods Enzymol. 1983;102:39–47. doi: 10.1016/s0076-6879(83)02006-6. [DOI] [PubMed] [Google Scholar]
  38. Watterson D. M., Iverson D. B., Van Eldik L. J. Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins. Biochemistry. 1980 Dec 9;19(25):5762–5768. doi: 10.1021/bi00566a015. [DOI] [PubMed] [Google Scholar]
  39. Yazawa M., Yagi K., Toda H., Kondo K., Narita K., Yamazaki R., Sobue K., Kakiuchi S., Nagao S., Nozawa Y. The amino acid sequence of the Tetrahymena calmodulin which specifically interacts with guanylate cyclase. Biochem Biophys Res Commun. 1981 Apr 30;99(4):1051–1057. doi: 10.1016/0006-291x(81)90725-7. [DOI] [PubMed] [Google Scholar]
  40. Zendegui J. G., Zielinski R. E., Watterson D. M., Van Eldik L. J. Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts. Mol Cell Biol. 1984 May;4(5):883–889. doi: 10.1128/mcb.4.5.883. [DOI] [PMC free article] [PubMed] [Google Scholar]

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