ANALYSIS OF BINDING OF BIOTINYLATED PROTOPLAST-RELEASE-INDUCING PROTEIN THAT INDUCES RELEASE OF GAMETIC PROTOPLASTS IN THE CLOSTERIUM-PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt+) cells and induces the release of gametic protoplasts from mating-type 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.