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Planta

, 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
Article

Abstract

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 

Abbreviations

ABC

a macromolecular complex of avidin DH and biotinylated horseradish peroxidase H

kDa

kilodalton

MIB medium

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

mt+

mating-type plus

mt

mating-type minus

PR-IP

protoplast-release-inducing protein

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

NHS-LC-biotin

sulfosuccinimidyl-6-(biotinamide) hexanoate

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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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