Fig. 4. The double and triple fusion-helix mutant minus gametes, fh1fh2 and fh1fh2fh3, express HAP2 on the cell surface at the mating structure, but fail to undergo fusion.
a Left panel: Structure of Chlamydomonas HAP2 showing the overall domain organization. The fusion helices are at the top of the structure. Right panel: Higher magnification view of the fusion helices with the hydrophobic residues in each of the helices that were mutated depicted in a stick format. (ii) Amino acid sequence of the HAP2 fusion surface with the residues mutated to alanine shown in red. b Neither the fh1fh2 nor the fh1fh2fh3 HAP2 mutants were capable of gamete fusion. WT and hap2 mutant minus gametes were mixed with an equal number of plus gametes for 10 min followed by assessment of gamete fusion. Results are averages of 4 independent experiments with 200 cells counted in each sample. c Protease-sensitivity assays showed that HAP2 in the fh1fh2, and fh1fh2fh3 minus gametes was located on the cell surface. Results are representative of 5 independent experiments. d Immunofluorescence of WT, fh1fh2, and fh1fh2fh3 minus gametes showed that the mutant HAP2s were localized similarly to WT. Scale bars are 5 m. e Mating structure adhesion assays of fh1fh2, and fh1fh2fh3 minus gametes (averages of at least 5 independent experiments with 200 cells counted in each sample. These assays were performed along with those of Fig. 2D, and have in common the WT x fus1 data). For b and e, The Kruskal-Wallis test and Dunn’s post-test were utilized to analyze the significance of differences with P values labeled above the groups compared. f Still images from a video (Supplementary Movie 2) of an fh1fh2fh3 fusion helix minus mutant undergoing mating structure attachment with a WT plus gamete. The adhesive interaction between the plus and minus mating structures, which appear as a single, thin strand (arrowheads) connecting the two gametes, resists the pulling forces generated by ciliary motility.