Sensitivity to low pH in nhx1Δ correlates with vacuolar hyperacidification, pointing to Na+(K+)/H+ antiport as a major leak pathway for exit of protons transported into the endosome/vacuole by the V-ATPase32. We evaluated vacuolar pH in situ by using the pH-sensitive fluorescent dye BCECF-AM, which is de-esterified and sequestered in the vacuolar lumen (Figure 3D, inset)38 allowing fluorescence intensity to be normalized to cell number (NI485) and calibrated to pH (Figure 3D). Vacuolar pH in vector-transformed nhx1Δ was more acidic relative to wild type, and similar to strains expressing the loss-of-function A438P and I222S variants. In contrast, vacuolar pH in cells expressing variants A438S, 1222L, and V167I was more alkaline, and similar to wild type Nhx1 (Figure 3E). A consequence of hyperacidic lumenal pH is that Vps10, a chaperone for lysosomal hydrolases, is retained and degraded in the prevacuolar compartment in 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
