Brett, Christopher L., Mark Donowitz, and Rajini Rao. Evolutionary origins of eukaryotic sodium/proton exchangers. Am J Physiol Cell Physiol 288: C223-C239, 2005; doi:10.1152/ajpcell.00360.2004.-More than 200 genes annotated as Na ϩ /H ϩ hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam. We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles. This analysis initially required examining the entire monovalent cation proton antiporter (CPA) superfamily that includes the CPA1, CPA2, and NaT-DC families of transporters, each of which has a unique set of bacterial ancestors. We have concluded that there are nine human NHE (or SLC9A) paralogs as well as two previously unknown human CPA2 genes, which we have named HsNHA1 and HsNHA2. The eukaryotic NHE family is composed of five phylogenetically distinct clades that differ in subcellular location, drug sensitivity, cation selectivity, and sequence length. The major subgroups are plasma membrane (recycling and resident) and intracellular (endosomal/TGN, NHE8-like, and plant vacuolar). HsNHE1, the first cloned eukaryotic NHE gene, belongs to the resident plasma membrane clade. The latter is the most recent to emerge, being found exclusively in vertebrates. In contrast, the intracellular clades are ubiquitously distributed and are likely precursors to the plasma membrane NHE. Yeast endosomal ScNHX1 was the first intracellular NHE to be described and is closely related to HsNHE6, HsNHE7, and HsNHE9 in humans. Our results link the appearance of NHE on the plasma membrane of animal cells to the use of the Na ϩ /K ϩ -ATPase to generate the membrane potential. These novel observations have allowed us to use comparative biology to predict physiological roles for the nine human NHE paralogs and to propose appropriate model organisms in which to study the unique properties of each NHE subclass. Naϩ /H ϩ exchanger; NHX; cation proton antiporter; phylogenetic analysis A BASIC PROPERTY OF LIFE is the ability of an organism to regulate cellular pH, volume, and ion composition. The transmembrane exchange of protons for sodium ions (Na ϩ ) is ubiquitous in organisms across all phyla and kingdoms, and underlies fundamental homeostatic mechanisms to control these ions. The family of Na ϩ /H ϩ exchangers (NHEs) plays an important role in diverse physiological processes, including control of cell cycle and cell proliferation (114, 117), transepithelial Na ϩ movement (174), salt tolerance (93, 130), vesicle trafficking, and biogenesis (5, 22). In mammals, NHE dysfunction is associated with pathophysiological conditions that include hypertension, epilepsy, postischemic myocardial arrhythmia, gastric and kidney disease, diarrhea, and glaucoma (36, 106, 174). Drugs such as S8218 and cariporide, which target specific NHE isoforms, are used to reduce the duration of apnea in animal studies and in clinical trials for the prevention of cardiac ischemia-reperfusion injur...

The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na ؉ /H ؉ exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome. Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases. Conversely, weak acids cause wild-type yeast to present nhx1⌬ trafficking phenotypes. Finally, we report that Nhx1 transports K ؉ in addition to Na ؉ , suggesting that a single mechanism may responsible for both pH and K ؉ -dependent endosomal processes. This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization. INTRODUCTIONIt is well established that luminal acidification of the endocytic pathway, including the endosome and lysosome/vacuole, is required for associated cellular function (Mellman et al., 1986;Mellman, 1992). Some examples include ligandreceptor dissociation and recycling of surface receptors, lysosome-mediated protein degradation, H ϩ -driven neurotransmitter loading and pH-dependent recycling of synaptic vesicles (Buckley et al., 2000;Nishi and Forgac, 2002). Similarly, viral pathogen entry and propagation is dependent on the pH gradient across the lumen of the endosome (Harley et al., 2001), and the abnormal lysosomal/endosomal morphologies and associated defective trafficking observed in a subset of lysosomal storage disorders are associated with abnormal changes in luminal pH (Futerman and van Meer, 2004). Pioneering experiments performed by Heuser clearly demonstrated that changes in cellular pH alone severely alter organellar morphology and movement (Heuser, 1989). This phenomenon can be explained by net changes in vesicle trafficking between compartments, as luminal pH can direct vesicle trafficking; thus, elevated pH in the endosome promotes endosome to Golgi vesicle movement (van Weert et al., 1995(van Weert et al., , 1997 also see Nieland et al., 2004). At the molecular level, local increases in pH are believed to be responsible for assembly of vesicle trafficking/sorting machinery in areas of the endosome destined for return to the plasma membrane (Maranda et al., 2001; also see Zeuzem et al., 1992;Aniento et al., 1996). Despite extensive evidence that changes in pH direct trafficking in this pathway, specific molecular mechanisms that control pH itself have not been defined. The ubiquitous Na ϩ /H ϩ exchangers of the NHE family are associated with cellular pH regulation (Orlowski and Grinstein, 2004). Recent phylogenetic analysis of the ...

SUMMARY Ca2+ is an essential and ubiquitous second messenger. Changes in cytosolic Ca2+ trigger events critical for tumorigenesis, such as cellular motility, proliferation and apoptosis. We show that an isoform of Secretory Pathway Ca2+-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuated basal Ca2+ levels and tumorigenicity. Contrary to its conventional role in Golgi Ca2+ sequestration, expression of SPCA2 increased Ca2+ influx by a mechanism dependent on the store-operated Ca2+ channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca2+ stores or STIM1 and STIM2 sensors, and uncoupled from Ca2+-ATPase activity of SPCA2. Binding of SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca2+ influx. Our findings reveal a signaling pathway in which Orai1-SPCA2 complex elicits constitutive store-independent Ca2+ signaling that promotes tumorigenesis.

Overexpression of the Arabidopsis thaliana vacuolar H ؉ -pyrophosphatase (AVP1) confers salt tolerance to the salt-sensitive ena1 mutant of Saccharomyces cerevisiae. Suppression of salt sensitivity requires two ion transporters, the Gef1 Cl ؊ channel and the Nhx1 Na ؉ ͞H ؉ exchanger. These two proteins colocalize to the prevacuolar compartment of yeast and are thought to be required for optimal acidification of this compartment. Overexpression of AtNHX1, the plant homologue of the yeast Na ؉ ͞H ؉ exchanger, suppresses some of the mutant phenotypes of the yeast nhx1 mutant. Moreover, the level of AtNHX1 mRNA in Arabidopsis is increased in the presence of NaCl. The regulation of AtNHX1 by NaCl and the ability of the plant gene to suppress the yeast nhx1 mutant suggest that the mechanism by which cations are detoxified in yeast and plants may be similar.

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca 2؉ -transporting ATPase in the Golgi, a hitherto unusual location for a Ca 2؉ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45 Ca 2؉ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca 2؉ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 3 Glu and Asp-371 3 Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H ؉ -ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca 2؉ ) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca 2؉ -ATPases. PMR1 therefore represents a new and distinct P-type Ca 2؉ -ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca 2؉ -ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.In eukaryotic cells, the vast bulk of cellular calcium is sequestered within intracellular calcium stores, which maintain cytoplasmic calcium ion concentrations at submicromolar levels and release calcium in response to physiological signals. A major intracellular calcium pool is the endoplasmic reticulum, which is well known for its prominent role in inositol 1,4,5-trisphosphate-and caffeine-mediated calcium release (2). Filling of this store is accomplished by a thapsigargin-sensitive Ca 2ϩ -ATPase, a member of the ubiquitous family of P-type ion pumps, and best characterized by the isoform in skeletal muscle sarcoplasmic reticulum (SERCA1 1 ; Refs. 3 and 4). There are data supporting the existence of another ionomycin-sensitive pool of intracellular calcium that is unresponsive to both inositol 1,4,5-trisphosphate and caffeine, is non-mitochondrial, and appears to be loaded by a thapsigargin-insensitive Ca 2ϩ

The internal environment of the ER is regulated to accommodate essential cellular processes, yet our understanding of this regulation remains incomplete. Cod1p/Spf1p belongs to the widely conserved, uncharacterized type V branch of P-type ATPases, a large family of ion pumps. Our previous work suggested Cod1p may function in the ER. Consistent with this hypothesis, we localized Cod1p to the ER membrane. The cod1Δ mutant disrupted cellular calcium homeostasis, causing increased transcription of calcium-regulated genes and a synergistic increase in cellular calcium when paired with disruption of the Golgi apparatus–localized Ca2+ pump Pmr1p. Deletion of COD1 also impaired ER function, causing constitutive activation of the unfolded protein response, hypersensitivity to the glycosylation inhibitor tunicamycin, and synthetic lethality with deletion of the unfolded protein response regulator HAC1. Expression of the Drosophila melanogaster homologue of Cod1p complemented the cod1Δ mutant. Finally, we demonstrated the ATPase activity of the purified protein. This study provides the first biochemical characterization of a type V P-type ATPase, implicates Cod1p in ER function and ion homeostasis, and indicates that these functions are conserved among Cod1p's metazoan homologues.

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma − phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Na؉ /H ؉ exchangers catalyze the electrically silent countertransport of Na ؉ and H ؉ , controlling the transmembrane movement of salt, water, and acid-base equivalents, and are therefore critical for Na ؉ tolerance, cell volume control, and pH regulation. In contrast to numerous well studied plasma membrane isoforms (NHE1-4), much less is known about intracellular Na ؉ /H ؉ exchangers, and thus far no vertebrate isoform has been shown to have an exclusively endosomal distribution. In this context, we show that the yeast NHE homologue, Nhx1 (Nass, R., Cunningham, K. W., and Rao, R. (1997) J. Biol. Chem. 272, 26145-26152), localizes uniquely to prevacuolar compartments, equivalent to late endosomes of animal cells. In living yeast, we show that these compartments closely abut the vacuolar membrane in a striking bipolar distribution, suggesting that vacuole biogenesis occurs at distinct sites. Nhx1 is the founding member of a newly emergent cluster of exchanger homologues, from yeasts, worms, and humans that may share a common intracellular localization. By compartmentalizing Na ؉ , intracellular exchangers play an important role in halotolerance; furthermore, we hypothesize that salt and water movement into vesicles may regulate vesicle volume and pH and thus contribute to vacuole biogenesis.