Alternative mechanisms of vacuolar acidification in H+-ATPase-deficient yeast

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proto n translocating ATPase (V-ATPase) is necessary for a variety of essential e ukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activit y (Delta vma) are viable when grown at low pH, suggesting alternative metho ds of organellar acidification. This was confirmed by directly measuring th e vacuolar pH by ratio fluorescence imaging. When Delta vma yeasts were cul tured and tested in the acidic conditions required for growth of V-ATPase-d eficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidi c extracellular medium cannot account for these observations, because the V -ATPase-independent vacuolar acidification was unaffected in mutants defici ent in endocytosis, Similarly, internalization of the plasmalemmal H+-ATPas e (Pma1p) was-ruled out, because overexpression of Pma1p failed to compleme nt the Delta vma phenotype and did not potentiate the vacuolar acidificatio n. To test whether weak electrolytes present in the culture medium could fe rry acid equivalents to the vacuole, wildtype and the Delta vma yeasts were subjected to sudden changes in extracellular pH, In both cell types, the v acuoles rapidly alkalinized when external pH was raised from 5.5 (the appro ximate pH of the culture medium) to 7.5 and re-acidified when the yeasts we re returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH4+, routinely added as a nitrogen source, was present . The NH4+-dependent acidification was not due to efflux of NH, from the va cuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH4+. Th ese findings suggest that although NH, can act as a cell-permeant proton sc avenger, NH4+ may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concent ration of NH4+ frequently added as a nitrogen source to yeast culture media together with effective NH4+ transporters thereby facilitate vacuolar acid ification when cells are suspended in acidic solutions.