Phenotype complementation in yeast offers a rapid and first approach towards functional screening of mutations. The nhx1Δ null strain exhibits clearly defined and quantifiable growth defects relating to pH, salt and drug sensitivity that have been linked to ion transport and vesicle trafficking32,33. Therefore, we introduced autism-associated mutations into equivalent positions in Nhx1 (Figure 2B; A438P, I222S and V167I). Because two of these positions carried moderate substitutions in Nhx1, we also generated “humanized” versions, A438S and I222L, equivalent to NHE9. All five substitutions and wild type Nhx1 were separately tagged with GFP or HA and expressed in nhx1Δ yeast. Like wild type Nhx1, most mutants were localized to 1–2 punctate compartments (Figure 2C) previously identified as prevacuolar endosomes38, and were expressed at equivalent levels (Figure 2D). Mutant A438P showed a shift in distribution to multiple puncta, suggesting a possible delay in trafficking of the mutant protein to the prevacuolar compartment.
