Novel Requirements for HAP2/GCS1-mediated Gamete Fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic D hap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2 , whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a singlepass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/ GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena's multiple mating type system.