Flagellar adhesion between mt(+) and mt(-) Chlamydomonas gametes regulatesphosphorylation of the mt(+)-specific homeodomain protein GSP1

During fertilization in Chlamydomonas, flagellar adhesion between mt(+) and mt(-) gametes induces a cAMP-dependent signal transduction pathway that pr epares the gametes for cell fusion and zygote formation. Previously, our la boratory identified a homeodomain protein (GSP1) whose expression was restr icted to the cell bodies of mt(+) gametes and whose transcript level was up regulated during flagellar adhesion. In this report, we describe a new form of GSP1 that appears early during gamete interactions, Immunoblot analysis showed that in addition to the 120-kDa form of GSP1 normally present in mt (+) gametes, a 122-kDa form was detected when the cells mere mixed with mt- gametes, The more slowly migrating form of GSP1 was detectable within minu tes after gametes were mixed together, and its appearance did not require n ew protein synthesis. Thus, the 122-kDa form represents a post-translationa l modification of the pre-existing 120-kDa form of GSP1. Moreover, conversi on to the 122-kDa form did not require cell fusion, Although the 120-kDa fo rm was expressed 10 h after vegetative cells were transferred to gametic in duction medium, the 122-kDa form was detected only after mt(+) gametes were induced to undergo the sexual signaling that accompanies fertilization. In cubation of mt(+) gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase in hibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 i mmunoprecipitated from signaling mt(+) gametes with alkaline phosphatase sh owed that the conversion was due to phosphorylation, The results indicate t hat flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of t he homeodomain protein GSP1 early during fertilization in Chlamydomonas.