nhx1Δ mutants. As a result, cargo destined for the vacuole, including carboxypeptidase Y (CPY), is missorted to the extracellular medium of nhx1Δ strains38, where it can be detected by Western analysis of slot blots (Figure 3F). Missorting of CPY to the medium was effectively rescued by plasmids expressing wild type Nhx1 or humanized variants A48S and I222L. In contrast, extracellular CPY was elevated in autism-associated variants A438P and I222S, similar to the vector-transformed nhx1Δ host strain. Again, variant V167I phenocopied wild type Nhx1 in rescuing missorting of CPY to the medium. Taken together, this analysis revealed that substitutions in the evolutionarily conserved sites on Nhx1, orthologous to autism associated variants S438P and L236S in NHE9, lead to loss of function, whereas a substitution in a variable region predicted to face the lipid bilayer, equivalent to V176I in NHE9, retained function in yeast Nhx1.
