The Functional Domain of GCS1-Based Gamete Fusion Resides in the Amino Terminus in Plant and Parasite Species

Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1), also called HAP2 (HAPLESS 2) in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga) and Plasmodium (malaria parasite) GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s) of GCS1, using complementation assay of ArabidopsisGCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite) knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.