Identification and characterization of a novel intracellular Na(+)/H(+) exchanger in Saccharomyces cerevisiae

All living cells maintain a relatively low intracellular Na+ concentration even in the presence of a large inwardly directed Na + gradient. The low intracellular Na+ concentrations are largely maintained by limiting the influx of Na+ into the cell, increasing the efflux of Na+ from the cell, and/or sequestering the Na+ in an intracellular compartment.; Three types of transport systems are believed to play integral roles in maintaining the low intracellular Na₊ concentrations. E1-E2 ATPases pump Na+ out of the cell against a large Na + gradient. Na+ symporters, which can transport Na + into the cell, limit Na+ influx in the presence of a large Na+ gradient by being differentially expressed and/or modulated. And Na+ antiporters, which utilize the electrochemical gradient on the plasma membrane or an intracellular compartment, transport Na+ out of the cell, or into an intracellular compartment. Although biochemical evidence existed for the presence of intracellular Na +/H+ exchangers in plants, yeast, and mammalian cells, prior to this work the identification of the exchanger at the molecular level was elusive.; This thesis describes a novel eukaryotic Na+/H + exchanger. It provides the first molecular identification of an intracellular Na+/H+ transporter, which we term NHX1, and shows that transport of Na+ into the prevacuolar compartment/late endosome contributes to Na+ tolerance in the yeast Saccharomyces cerevisiae. This thesis also reports that NHX1 is required for Na+ sequestration and contributes to the Na+ tolerant phenotype of a plasma membrane H +-ATPase (pma1) mutant. Furthermore, these studies also provide evidence that NHX1 is induced upon culturing in NaCl, and contributes to osmotolerance. Finally, these studies show that NHE6, a newly discovered human Na+/H+ exchanger highly homologous to NHX1, confers Na+ tolerance in yeast and may also be intracellularly localized.