Flagellar adhesion between mt+ and mt- Chlamydomonas gametes regulates phosphorylation of the mt+ -specific homeodomain protein GSP1

• Published in: The Journal of biological chemistry 1999-11, Vol.274 (48), p.34383-34388
• Main Authors: Wilson, N.F, O'Connell, J.S, Lu, M, Snell, W.J
• Format: Article
• Language: English
• Subjects: adhesion; Animals; Cell Adhesion - genetics; Cell Adhesion - physiology; Chlamydomonas reinhardtii; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - growth & development; Chlamydomonas reinhardtii - metabolism; cyclic AMP; Cyclic AMP - physiology; Electrophoresis, Polyacrylamide Gel; fertilization (reproduction); Flagella - physiology; flagellum; germ cells; Homeodomain Proteins - chemistry; Homeodomain Proteins - metabolism; Immunoblotting; Molecular Weight; Mutation; Phosphorylation; Plant Proteins; proteins; Reproduction - genetics; Signal Transduction; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.48.34383

During fertilization in Chlamydomonas, flagellar adhesion between mt(+) and mt(-) gametes induces a cAMP-dependent signal transduction pathway that prepares the gametes for cell fusion and zygote formation. Previously, our laboratory identified a homeodomain protein (GSP1) whose expression was restricted 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 were mixed with mt(-) gametes. The more slowly migrating form of GSP1 was detectable within minutes after gametes were mixed together, and its appearance did not require new protein synthesis. Thus, the 122-kDa form represents a post-translational modification of the pre-existing 120-kDa form of GSP1. Moreover, conversion to the 122-kDa form did not require cell fusion. Although the 120-kDa form was expressed 10 h after vegetative cells were transferred to gametic induction medium, the 122-kDa form was detected only after mt(+) gametes were induced to undergo the sexual signaling that accompanies fertilization. Incubation of mt(+) gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase inhibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 immunoprecipitated from signaling mt(+) gametes with alkaline phosphatase showed that the conversion was due to phosphorylation. The results indicate that flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of the homeodomain protein GSP1 early during fertilization in Chlamydomonas.