Sensitivity to hygromycin B toxicity is increased in nhx1Δ as a result of defective trafficking to the vacuole, believed to be the site of drug detoxification32,33. Plasmid expressing wild type Nhx1 conferred tolerance to hygromycin B growth toxicity (Figure 3A). Similarly, the humanized versions of Nhx1, A438S and I222L, were equally effective in protecting against drug toxicity. In contrast, two autism-associated variants, A438P and I222S, closely resembled the vector-transformed null mutant, consistent with complete loss of function. The third variant, V167I, resembled wild type Nhx1. We obtained similar results with growth sensitivity to high salt (Figure 3B) and to low pH (Figure 3C). In each case, the control substitutions, equivalent to those in NHE9, resembled wild type, whereas autism associated variants A438P and I222S were similar to the vector-transformed nhx1Δ null strain. Consistently, the V167I variant showed no loss of function in these growth phenotypes.
