We generated a deep alignment of 230 Ire1 orthologs from fungi (211), representative plants (9) and animals (10). We compared residues at three positions, which correspond to the S. cerevisiae Ire1 E988, K992, and Y1059. Among the 230 orthologs being analyzed, 175 of them have the S. pombe-like pattern (blue)—with a varying amino acid at position 988, a negatively charged amino acid (aspartate or glutamate) at position 992 and a positively charged amino acid (lysine or arginine) at position 1059. Thirty-three of the Ire1 orthologs have the S. cerevisiae-like pattern—with a negatively charged amino acid (aspartate or glutamate) at position 988, a positively charged amino acid (lysine or arginine) at position 992 and mostly tyrosine at position 1059. Tyrosine at position 1059 may orchestrate the positioning of lysine/arginine side chain at position 992 (Lee et al., 2008). The S. cerevisiae-like pattern is restricted to the Saccharomycotina. In 6 Ire1 orthologs, the S. cerevisiae- or S. pombe-like inter-molecular salt bridge are replaced with a putative hydrogen bond (marked with x). Ire1 orthologs from higher plants exhibit a pattern distinct from S. cerevisiae and S. pombe. One interesting case (Table insert A) is with the Ire1 from Yarrowia lipolytica, whose location on the evolutionary tree is at the boundary between S. cerevisiae-like and S. pombe-like species. Yarrowia lipolytica Ire1 is in a transitioning state from the S. cerevisiae-like into the S. pombe-like Ire1—its amino acids at position 988 (aspartate) and 1059 (tyrosine) remain S. cerevisiae-like whereas its amino acid at position 992 (glutamate) changed from S. cerevisiae-like (positively charged) into S. pombe-like (negatively charged). The case of Yarrowia lipolytica Ire1 suggests that salt-bridge rewiring occurs during the evolutionary process. Another interesting case (Table insert B) is with the Ire1 from Capronia epimyces. While its neighboring species have S. pombe-like pattern, Capronia epimyces Ire1 undergoes a unique evolution with arginine, aspartate and methionine at positions 988, 992, and 1059, making it a charge reversal (at positions 988 and 992) of S. cerevisiae-like Ire1. The detailed sequences are listed in Figure 5—source data 1.