Fig. 5. Fusion helix membrane interaction made possible by mating structure attachment promotes trimer formation and trimer formation ceases when adhesion ceases.

a, b Semi-native SDS-PAGE immunoblots showing HAP2 trimer formation in fusion helix mutant minus gametes after mixing with plus gametes for the times indicated. c The fraction of surface HAP2 forming trimers was strongly reduced in fh1fh2 and fh1fh2fh3 compared to fh1 gametes after mixing with WT plus gametes. Signal intensities of the trimer band and the upper monomer band at 5 min time points from at least three independent experiments were quantified by Image Studio Lite (LI-COR Biosciences). The Kruskal-Wallis test and Dunn’s post-test were utilized to analyze the statistical differences among mutants. P-values are indicated above the corresponding columns. d fh1 minus gametes mixed with WT plus gametes failed to undergo hemifusion. WT plus gametes were incubated with membrane dye PKH26, washed into N-free medium, and mixed with the indicated minus gamete mutants for at least 20 min. Left panels are fluorescence images and right panels are phase-contrast images. At least 200 pairs of cells were observed with similar results. Scale bars, 5 μm. e Semi-native SDS-PAGE immunoblot showing HAP2 trimer formation in WT minus gametes after mixing with WT plus gametes for the indicated times. % fusion is shown under the lanes. The blot images in a, b, and e are representative of at least 3 independent experiments.