Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast

Abstract

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (VATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking VATPase activity (⌬vma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When ⌬vma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H ؉ -ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the ⌬vma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wildtype and the ⌬vma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH 4 ؉ , routinely added as a nitrogen source, was present. The NH 4 ؉ -dependent acidification was not due to efflux of NH 3 from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH 4 ؉ . These findings suggest that although NH 3 can act as a cell-permeant proton scavenger, NH 4 ؉ may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH 4 ؉ frequently added as a nitrogen source to yeast culture media together with effective NH 4 ؉ transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.Acidification of defined endomembrane compartments along the endocytic and secretory pathways is essential for cellular function. Intraorganellar acidification appears to control vesicular traffic (1) as well as receptor ligand dissociation within endosomes (1). Similarly, acidification is thought to be essential for protein degradation in lysosomes (2) and for microbial elimination in phagosomes (3). In yeast and plants acidification of the vacuole provides the driving force for secondary transport of a variety of ions and metabolites (4 -7).Acid equivalents are concentrated in the lumen of these organelles by active H ϩ pumping mediated by the vacuolartype ATPase (V-ATPase), 1 an evolutionarily conserved multimeric enzyme (see Refs. 8 -11 for review). Considering the array of critical cellular functions that depend on the acidification generated by the V-ATPase, this enzyme would be predicted to be e...

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

“…V-ATPase-independent vacuolar acidification has been observed previously and attributed to partitioning of weak electrolytes (16,32). V-ATPaseindependent acidification of prevacuolar compartments in the vph1⌬ and vma2⌬ mutant could result from similar mechanisms or from various H ϩ antiporters able to transport H ϩ into organelles under the more favorable pH gradients provided by cytosolic acidification.…”

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase

“…The impaired acidification of vacuoles is thought to be one of the major causes of the growth inhibition of V-ATPase-deficient mutants at neutral pH (Li & Kane, 2009). Even in wild-type cells, the vacuolar pH can vary slightly depending on the extracellular pH; that is, the vacuolar pH increases from approximately 5.5 to 5.9 when the pH of the culture medium is switched from 5.5 to 7.5 (Plant et al, 1999). Thus, it is likely that the increase in the MIPC levels is induced by the impaired acidification of vacuoles.…”

“…3a, b). In V-ATPase-deficient mutants, the vacuolar pH increases with increasing extracellular pH; that is, the vacuolar pH is approximately 5.9 when the cells are cultured at pH 5.5, whereas it increases to approximately 7 when the pH of culture medium is switched to 7.5 (Plant et al, 1999). The impaired acidification of vacuoles is thought to be one of the major causes of the growth inhibition of V-ATPase-deficient mutants at neutral pH (Li & Kane, 2009).…”

Ratiometric Imaging of pH Probes

“…For example, increased cytosolic pH and decreased extracellular pH at the leading edge of migrating cells are evolutionarily conserved signals necessary for directed cell migration. Experiments using BCECF and SNARF demonstrate that increased pHi promotes de novo actin polymerization in a variety of organisms including sea urchin eggs (Begg & Rebhun, 1979), the slime mold Dictyostelium discoideum (Patel & Barber, 2005;Plant et al, 1999;Van Duijn & Inouye, 1991), and mammalian cells (Denker & Barber, 2002). More recently, the use of TIRF microscopy using BCECF (Brett et al, 2005;Ludwig, Schwab, & Stock, 2013) and paxillin-mCherry-pHluorin ( Fig.…”

“…As described in the preceding text, cleavage of the methyl ester of the AM forms of these dyes is dependent on the activity of intracellular esterases, which can lead to dye accumulation in organelles with high esterase activity. For example, BCECF-AM cannot be used to measure cytosolic pH in the yeast Saccharomyces cerevisiae because it accumulates in the yeast vacuole instead of the cytosol, in contrast to cytosolic accumulation in mammalian cells (Plant, Manolson, Grinstein, & Demaurex, 1999). For yeast cells, the genetically encoded pH sensor pHluorin, described in the succeeding text, is more effective for measuring pHi (Brett, Tukaye, Mukherjee, & Rao, 2005).…”