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J Biol Chem, Vol. 274, Issue 52, 37270-37279, December 24, 1999
From the Acidification of the
endosomal/lysosomal pathway by the vacuolar-type proton translocating
ATPase (V-ATPase) is necessary for a variety of essential eukaryotic
cellular functions. Nevertheless, yeasts lacking V-ATPase activity
(
Alternative Mechanisms of Vacuolar Acidification in
H+-ATPase-deficient Yeast*
§,§,§¶, and
**
Division of Cell Biology, Hospital for Sick Children,
Toronto, Ontario M5G 1X8, the § Department of Biochemistry,
University of Toronto, Toronto, Ontario M5S 2Z9, Canada, and the
Department of Physiology, University of Geneva Medical
Center, 1211 Geneva 4, Switzerland
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, wild-type 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
NH4+,
routinely added as a nitrogen source, was present. The
NH4+-dependent
acidification was not due to efflux of NH3 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
NH4+. These
findings suggest that although NH3 can act as a
cell-permeant proton scavenger,
NH4+ may
function as a protonophore, facilitating equilibration of the pH across
the plasma and vacuolar membranes of yeast. The high concentration of
NH4+
frequently added as a nitrogen source to yeast culture media together with effective
NH4+
transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.
*
This work was supported by operating grants from the Medical
Research Council of Canada (to S. G. and M. M.) and by Grant 31-46859.96 from the Swiss National Science Foundation (to N. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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