Fig. 3

Human and zebrafish IZUMO1, SPACA6, and TMEM81 interact (A) Relative protein levels in wild-type and mutant zebrafish sperm by PRM-MS42 normalized to 6 constant proteins. (B) Volcano plots of differentially enriched proteins in FLAG coIPs from gently fixed sperm expressing Tmem81-3xFLAG-sfGFP (left) or Izumo1-3xFLAG-sfGFP (right) versus wild-type sperm. Trimer members are highlighted. False discovery rate (FDR) thresholds43 indicated as dotted (1% FDR) and dashed lines (5% FDR). (C) Complex formation between Izumo1, Spaca6, and Tmem81 is required for male fertility in zebrafish. Left: zoomed-in views of the predicted interfaces between zebrafish Izumo1 (yellow), Spaca6 (blue), and Tmem81 (red) with mutated amino acids shown as sticks as modeled by AlphaFold-Multimer. Right: fertilization rates in zebrafish of transgenic males in the respective mutant background with wild-type females. Fertilization rates for tmem81−/−, tg[tmem81], izumo1−/−, and tg[izumo1] replotted from Figure 2A. (D) Human orthologs interact in vitro. Freestyle 293-F cells were transfected to co-express full-length human sperm trimer proteins and induced to shed giant plasma membrane vesicles (GPMVs)44 with N-ethylmaleimide (NEM) and Ca2+. n-Dodecyl-β-D-maltoside (DDM)-solubilized GPMVs were used for coIP. Western blot of FLAG coIP after co-expression of Hs IZUMO1-3xFLAG, Hs SPACA6-3xV5, and Hs TMEM81-ALFA or the individual proteins. The plasma membrane protein Na+/K+ ATPase controls for non-specific capture of membrane fragments. Bar graphs represent mean ± SD. Significant p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons test. All other comparisons are non-significant. See also Figure S4.