Fig. 4: Evolution of MCU–EMRE interaction.

a, b Phylogenetic trees of members of MCU (a) and EMRE (b) protein families, and sequence diversity of major domains. The sequence alignment of TM1 and TM2 of MCU sequences from 20 species is shown in a. The program Multi-Harmony⁴³ was used to detect residues that are overall conserved but differ in the MCUP members (highest scoring positions are indicated with red arrows). The MCUP clade is shown in purple. The evolutionary point where the MCU proteins become EMRE dependent is shown in gray. The degree of conservation across the animal-related MCU members is very high in these loci, while few positions are Holomycota or Holozoa specific. Similarly, in b EMRE’s sequence diversity across opisthokonts is shown for the β-hairpin, the TM, and CAD domain. Residues found important for the interaction between MCU and EMRE in ref. ²⁰ and fully conserved positions are indicated with gray and red arrows, respectively. c–f Representative traces and quantification of mt-Ca²⁺ transients in yeast cells expressing either human and animal-like S. punctatus and A. macrogynus MCU with human EMRE (n = 3) (c), species-specific EMRE with human MCU (n = 3) (d), animal-like S. punctatus and A. macrogynus MCU with their respective wild type or truncated (-t) EMREs (n = 4) (e) or with human EMRE fused to the chytrid extra C-terminal domain (n = 4) (f) upon glucose-induced calcium (GIC) stimulation in the presence of 1 mM CaCl₂. All data represent mean ± SEM. P values are indicated in the different panels (c, e, f ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test; d *p = 0.012, one-way ANOVA with Dunnett’s multiple comparisons test). Source data for c–f are provided as a Source Data file.