Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast: THE ENDOSOME/PREVACUOLAR COMPARTMENT IS A TARGET FOR SALT TOXICITY*

Mutants of the plant cation/H⁺ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by ³¹P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H⁺ antiporter.The Arabidopsis thaliana vacuolar alkali cation transporter AtNHX1 has been shown to increase salt tolerance in transgenic plants of several species (1). In Saccharomyces cerevisiae, its ortholog (ScNHX1) is mainly localized in late endosomes, where it is thought to contribute to vacuole biogenesis by regulating pH and vesicle volume (2). ScNHX1 itself has a role in halotolerance. Deletion of ScNHX1 confers salt sensitivity and diminishes Na⁺ compartmentalization, albeit indirectly, since the unrelated VNX1 exchanger accounts for most of the cation/H⁺ antiport activity in the tonoplast of yeast (3, 4). However, AtNHX1 complements a yeast mutant defective in ScNHX1 and restores cation compartmentalization (5).Improving the salt tolerance of crop plants is an important goal in biotechnology. In addition to the mechanisms by which a cell can cope with increased concentrations of toxic cations, it is important to know the identity of salt-sensitive cellular targets. Only a few key processes have been identified. In yeast, HAL2, an inositol phosphatase that catalyzes the dephosphorylation of 3′-phosphoadenosine-5′-phosphate to AMP, has been found to be inhibited by Li⁺ and Na⁺. Inhibition of HAL2 during salt stress results in the accumulation of 3′-phosphoadenosine-5′-phosphate in the cell, which has the potential to produce a variety of toxic effects, such as the inhibition of sulfotransferases and RNA-processing enzymes (6). Another possible target is the KEX2/furin family of proteases of the Golgi/secretory pathway. The activity of KEX2 in vitro has been shown to respond differently, depending on the alkali cation and concentration present in the medium (7). Here, we show that the endosomal system is an additional target for Na⁺ toxicity.The Golgi apparatus, trans-Golgi network, and endosome/prevacuolar compartment form a continuum where proteins and membranes are modified en route to their final destinations (8–10). The late endosome/prevacuolar compartment is considered a key point in intracellular vesicle and protein trafficking. In addition to being the previous stage for vacuolar sorted proteins and cargo, this is where both the exocytic and endocytic pathways converge (10, 11). Ion homeostasis in these organelles is increasingly regarded as an important feature for intracellular transport processes (12–15). In particular, K⁺ concentration may regulate the activity and specificity of enzymes modifying proteins posttranslationally, such as the above mentioned KEX2/furin protease family (7). Lumenal pH has been reported also to regulate selective protein aggregation in secretory vesicles (12). In this respect, it is noteworthy that yeast nhx1 mutants have been characterized as class E vps mutants with impaired vacuole biogenesis and protein sorting (15).AtNHX1 is thought to increase salt tolerance in plants through the intracellular compartmentation of Na⁺. However, using purified protein, it has been shown that this antiporter can exchange H⁺ for K⁺, Na⁺, or Li⁺, albeit the last one with lower affinity (16). The poor K⁺/Na⁺ selectivity raises the question of whether Na⁺ transport is the primary function of AtNHX1 in plant cells and if AtNHX1 is amenable to selection of better alleles for salt tolerance. Mutagenesis of cation transporters has proved to be a valuable tool to obtain alleles with modified transport activities (17, 18). At the same time, this provides information about the important amino acid residues that affect the mechanism of protein function. In this work, we sought to produce hypermorphic AtNHX1 alleles conferring greater salt tolerance, by either improved Na⁺/K⁺ discrimination or altered protein regulation. We show here that nhx1-deficient yeast cells that express mutated forms of AtNHX1 display improved halotolerance compared with cells that express the wild-type AtNHX1. The mutations responsible for these changes were scattered throughout the hydrophobic N terminus of the protein, and their effect was to introduce bulkier side chain amino acids. Surprisingly, the result of these mutations was not increased compartmentalization of toxic alkali cations. Instead, all of these mutants showed a decreased content of Li⁺ (a tracer for Na⁺), whereas full amounts of K⁺ were retained. Biochemical characterization of a selected mutant transporter showed greater cation discrimination favoring K⁺ transport. AtNHX1 is localized to the vacuole and late endosome/prevacuolar compartment. Together, these results suggest that the endomembrane system is a cellular target of Na⁺ intoxication.