, Volume 189, Issue 3, pp 468–474 | Cite as

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

  • Hiroyuki Sekimoto
  • Shinobu Satoh
  • Tadashi Fujii


A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt+) cells and induces the release of gametic protoplasts from matingtype minus (mt) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10−8 M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.

Key words

Closterium Conjugation (ClosteriumProtoplast-release Inducing protein Pheromone Receptor 



a macromolecular complex of avidin DH and biotinylated horseradish peroxidase H



MIB medium

MI medium containing 1% (w/v) bovine serum albumin


mating-type plus


mating-type minus


protoplast-release-inducing protein


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


sulfosuccinimidyl-6-(biotinamide) hexanoate


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  1. Adair, W.S. (1985) Characterization of Chlamydomonas sexual agglutinins. J. Cell Sci. Suppl. 2, 233–260PubMedGoogle Scholar
  2. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254PubMedCrossRefGoogle Scholar
  3. Cook, P.A. (1963) Variation in vegetative and sexual morphology among the small curved species of Closterium. Phycologia 3, 1–18Google Scholar
  4. Dubois-Tylski, T. (1973) La conjugation en culture ‘in vitro’ chez Closterium rostratum. Eur. Bull. Soc. Bot. Fr. 120, 33–41Google Scholar
  5. Gershoni, J.M., Palade, G.E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124, 396–405PubMedCrossRefGoogle Scholar
  6. Gilles, R., Gilles, C., Jaenicke, L. (1984) Pheromone-binding and matrix-mediated events in sexual induction of Volvox carteri. Z. Naturforsch. 39c, 584–592Google Scholar
  7. Hamada, J. (1978) Studies on several environmental factors for zygote formation and germination in Closterium ehrenbergii. Bot. Mag. Tokyo 91, 173–180Google Scholar
  8. Hamada, J., Yoshizawa-Katoh, T., Tsunewaki, K. (1982) Genetic study on mating type genes by a new type of tetrad analysis in Closterium ehrenbergii. Bot. Mag. Tokyo 95, 101–108Google Scholar
  9. Hedo, J.A., Simpson, I.A. (1984) Internalization of insulin receptors in the isolated rat adipose cell. J. Biol. Chem. 259, 11083–11089PubMedGoogle Scholar
  10. Hogetsu, T., Yokoyama, M. (1979) Light, a nitrogen-depleted medium and cell-cell interaction in the conjugation process of Closterium ehrenbergii Meneghini. Plant Cell Physiol. 20, 811–817Google Scholar
  11. Ichimura, T. (1971) Sexual cell division and conjugation-papilla formation in sexual reproduction of Closterium strigosum. In: Proc. of the 7th Int. Seaweed Symp, pp. 208–214, Nishizawa, K., ed. University of Tokyo Press, TokyoGoogle Scholar
  12. Ichimura, T. (1983) Hybrid in viability and predominant survival of mating type minus progeny in laboratory crosses between two closely related mating groups of Closterium ehrenbergii. Evolution 37, 252–260CrossRefGoogle Scholar
  13. Ichimura, T., Kasai, F. (1987) Time-lapse analysis of sexual isolation between two closely related mating groups of the Closterium ehrenbergii species complex (Chlorophyta). J. Phycol. 23, 523–534CrossRefGoogle Scholar
  14. Kasai, F., Ichimura, T. (1986) Morphological variabilities of three closely related groups of Closterium ehrenbergii Meneghini (Chlorophyta). J. Phycol. 22, 158–168Google Scholar
  15. Kasai, F., Ichimura, T. (1987) Stable diploids from intragroup zygospores of Closterium ehrenbergii menegh. (Conjugatophyceae). J. Phycol. 23, 344–351Google Scholar
  16. Kasai, F., Ichimura, T. (1990) A sex determining mechanism in the Closterium ehrenbergii (Chlorophyta) species complex. J. Phycol. 26, 195–201CrossRefGoogle Scholar
  17. Kato, A., Sasaki, K. (1983) Effect of tunicamycin on sexual reproduction in heterothallic strains of Closterium. J. Fac. Sci. Hokkaido Univ. Ser. V 13, 1–6Google Scholar
  18. Kato, A., Sasaki, K. (1985) Sexual interaction in heterothallic strains of Closterium peracerosum-strigosum-littorale. Plant Physiol. 77, 556–559PubMedGoogle Scholar
  19. Kato, A., Obokata, J., Sasaki, K. (1981) Mating type interaction in Closterium peracerosum-strigosum-littorale: mating induced protoplast release. Plant Cell Physiol. 22, 1215–1222Google Scholar
  20. Kato, A., Takagi, T., Sasaki, K. (1983) Light conditions for sexual reproduction in heterothallic strains of Closterium. Plant Cell Physiol. 24, 93–100Google Scholar
  21. Karlson, P., Luscher, M. (1959) ‘Pheromones’: A new term for a class of biologically active substances. Nature 183, 55–56PubMedCrossRefGoogle Scholar
  22. Kochert, G. (1981) Sexual pheromones in Volvox development. In: Sexual interaction in eukaryotic microbes, pp. 73–93, O'Day, D.H., Horgen, P.A., eds. Academic Press, New YorkGoogle Scholar
  23. Kozaki, A., Takeba, G. Tanaka, O. (1991) A polypeptide that induces flowering in Lemna paucicostata at a very low concentration. Plant Physiol. 95, 1288–1290PubMedCrossRefGoogle Scholar
  24. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685PubMedCrossRefGoogle Scholar
  25. Lippert, B.E. (1967) Sexual reproduction in Closterium moniliferum and C. ehrenbergii. J. Phycol. 3, 182–198Google Scholar
  26. Mages, H.W., Tschochner, H., Sumper, M. (1988) The sexual inducer of Volvox carteri — Primary structure deduced from cDNA sequence. FEBS Lett. 234, 407–410PubMedCrossRefGoogle Scholar
  27. Pearce, G., Strydom, D., Johnson, S., Ryan, C.A. (1991) A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253, 895–898PubMedGoogle Scholar
  28. Pickett-Heaps, J.D., Fowke, L.C. (1971) Conjugation in the desmid Closterium littorale. J. Phycol. 7, 37–50CrossRefGoogle Scholar
  29. Scatchard, G. (1948) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660–672Google Scholar
  30. Sekimoto, H., Fujii, T. (1992) Analysis of gametic protoplastrelease in Closterium peracerosum-strigosum-littorale complex (Chlorophyta). J. Phycol. 28, 615–619CrossRefGoogle Scholar
  31. Sekimoto, H., Satoh, S., Fujii, T. (1990) Biochemical and physiological properties of a protein inducing protoplast release during conjugation in the Closterium peracerosum-strigosum-littorale complex. Planta 182, 348–354CrossRefGoogle Scholar
  32. Snell, W.J. (1985) Cell-cell interactions in Chlamydomonas. Annu. Rev. Plant Physiol. 36, 287–315CrossRefGoogle Scholar
  33. Takeba, G., Nakajima, Y., Kozaki, A., Tanaka, O., Kasai, Z. (1990) A flower-inducing substance of high molecular weight from higher plants. Plant Physiol. 94, 1677–1681PubMedGoogle Scholar
  34. Tschochner, H., Lottspeich, F., Sumper, M. (1987) The sexual inducer of Volvox carteri: purification, chemical characterization and identification of its gene. EMBO J. 6, 2203–2207PubMedGoogle Scholar
  35. Watanabe, M.M., Ichimura, T. (1978a) Biosystematic studies of the Closterium peracerosum-strigosum-littorale complex II. Reproductive isolation and morphological variation among several populations from the Northern Kanto area in Japan. Bot. Mag. Tokyo 91, 1–10Google Scholar
  36. Watanabe, M.M., Ichimura, T. (1978b) Biosystematic studies of the Closterium peracerosum-strigosum-littorale complex III. Degree of sexual isolation among the three population groups from the Northern Kanto area. Bot. Mag. Tokyo 91, 11–24Google Scholar
  37. Watanabe, M.M., Ichimura, T. (1982) Biosystematic studies of the Closterium peracerosum-strigosum-littorale complex IV. Hybrid breakdown between two closely related groups, Group II-A and Group II-B. Bot. Mag. Tokyo 95, 241–247Google Scholar
  38. Wiese, L. (1984) Mating systems in unicellular algae. In: Encyclopedia of plant physiology, N. S. vol. 17: Cellular interaction, pp. 238–260, Linskens, H.F., Heslop-Harrison, J., eds. Springer, Berlin Heidelberg New York TokyoGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Hiroyuki Sekimoto
    • 1
  • Shinobu Satoh
    • 1
  • Tadashi Fujii
    • 1
  1. 1.Institute of Biological SciencesUniversity of TsukubaIbarakiJapan

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