Flagellar adhesion between mt(+) and mt(-) Chlamydomonas gametes regulates phosphorylation 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 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.
Wilson
N F
NF
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039, USA.
O'Connell
J S
JS
Lu
M
M
Snell
W J
WJ
D000818:Animals; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000502:physiology*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008970:Molecular Weight; D009154:Mutation; D010766:Phosphorylation; D010940:Plant Proteins; D012098:Reproduction / Q000235:genetics; D015398:Signal Transduction; D013997:Time Factors
C116848:GSP1 protein, Chlamydomonas; D018398:Homeodomain Proteins; D010940:Plant Proteins; D000242:Cyclic AMP