Novel Localization of a Na+/H+ Exchanger in a Late Endosomal Compartment of Yeast

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. 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. Na+/H+ exchangers of eukaryotic cells comprise a family of membrane proteins catalyzing the electroneutral countertransport of Na+ and H+ (1Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (563) Google Scholar, 2Tse C.-M. Levine S.A. Brant S.R. Nath S. Pouyssegur J. Donowitz M. Cell. Physiol. Biochem. 1994; 4: 282-300Crossref Scopus (37) Google Scholar, 3Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). At the plasma membrane of animal cells, the prevailing Na+gradient generated by the Na+/K+-ATPase is used to drive H+ equivalents from the cell. As such, these exchangers are involved in the regulation of intracellular pH, cell volume control, and transcellular Na+ movements in epithelial tissue. These functions are closely related to physiological and pathophysiological cellular events, including fertilization, cell cycle control, differentiation, essential hypertension, gastric and kidney disease, and epilepsies. Na+/H+ exchange activity has been detected in virtually every cell type that has been examined, and at least six distinct NHE isoforms have been identified thus far. Molecular cloning of the first Na+/H+exchanger (4Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar) led to a predicted membrane topology based on the hydropathy profile of the amino acid sequence: there are 12 membrane-spanning segments comprising a discrete N-terminal structural domain of approximately 500 residues, followed by a long cytoplasmic C-terminal tail of approximately 300 residues. This predicted structural subdivision mimics a partition of function: analysis of deletion mutants has shown that the membrane-embedded domain retains the ability to insert into the plasma membrane, is transport-competent, and is sensitive to inhibition by amiloride and its derivatives (5Wakabayashi S. Fafournoux P. Sardet C. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (238) Google Scholar). However, it is the C-terminal domain that carries multiple protein kinase consensus sites, binds calmodulin, and mediates the response to a multitude of regulatory signals involved in control of cell proliferation, volume, and osmolarity changes. All Na+/H+ exchangers that have been characterized at a molecular level thus far localize predominantly, if not exclusively, to the plasma membrane. Nevertheless, there has been biochemical documentation of Na+/H+ exchange activity in endosomal preparations from kidney, liver, zymogen granules of pancreatic acinar cells, and chromaffin granules of adrenal glands (6Gurich R.W. Warnock D.G. Am. J. Physiol. 1986; 251: F702-F709PubMed Google Scholar, 7Hilden S.A. Ghoshroy K.B. Madias N.E. Am. J. Physiol. 1990; 258: F1311-F1319PubMed Google Scholar, 8Hensley C. Bradley M. Mircheff A.K. Kidney Int. 1990; 37: 707-716Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 9Van Dyke R.W. Am. J. Physiol. 1995; 269: C943-C954Crossref PubMed Google Scholar, 10Thevenod A.F. J. Membr. Biol. 1996; 152: 195-205Crossref PubMed Scopus (21) Google Scholar, 11Haigh J.R. Phillips J.H. Biochem. J. 1989; 257: 499-507Crossref PubMed Scopus (17) Google Scholar). In each case, the exchange activity was reported to coexist with a distinct subset (∼20%) of vesicles containing the vacuolar H+-ATPase and to exhibit kinetic similarity with the plasma membrane exchangers with respect to reversibility, simple hyperbolic response to Na+, and allosteric activation by H+. However, amiloride did not inhibit the endocytic exchange activity, Li+ was a poor substrate but a good inhibitor of Na+/H+ exchange, and theKm for Na+ was somewhat lower than that seen for plasma membrane isoforms (4.7–10 mm versus 15–18 mm), suggesting that the endocytic exchanger is a distinct molecular isoform. In earlier work, we have shown that the NHX1 gene ofSaccharomyces cerevisiae mediates sequestration of Na+ within an intracellular compartment, suggestive of a novel intracellular localization (12Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Here, we provide direct evidence that Nhx1 localizes exclusively to a unique late endosomal compartment, thus providing a starting point to explore the molecular, cellular, and physiological functioning of a completely novel member of this family of transporters. We have also observed the emergence of new exchanger homologues in other organisms, as a result of systematic sequencing efforts worldwide, that share greater homology with yeast Nhx1 than to the plasma membrane isoforms. We suggest that the sequence similarites among these newly discovered isoforms is indicative of a common intracellular, possibly endosomal localization. Strains K601 (wild type) and R100 (Δnhx1) used in this study are isogenic to W303 and have been described (12Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). A 4.5-kilobase pair (kbp) 1The abbreviations used are: kbpkilobase pairPCRpolymerase chain reactionHAhemagglutininGFPgreen fluorescent proteinPVCprevacuolar compartment. SalI insert containing the intact NHX1 gene was recovered from cosmid C9410 (American Type Culture Collection), and a 3-kbp SalI to SpeI fragment from the 5′ portion of the gene was cloned into pRS425 (13Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene ( Amst. ). 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar), now called pRin72. The C terminus of Nhx1 was tagged with a triple hemagglutinin (HA) epitope using two polymerase chain reaction (PCR) products as primers for a third PCR. The following primers were used to amplify a 0.8-kbp product from cosmid C9410 that extended from +1.1 kbp downstream from the initiating ATG to the end of the NHX1 open reading frame, with the removal of the termination codon, and addition of a NotI site and a short sequence homologous to the 5′ end of the HA epitope: 5′-CTGAAGTAGAACTAGTCTATAAGCCAC-3′ (sense) and 5′-AACATCGTATGGGTAAAAGATGCGGCCGCCGTGGTTTTGGGAAGAGAAATCTGCAGG-3′ (antisense). The second PCR created a 1-kbp product beginning with a short sequence homologous to the 3′ end of the HA epitope, followed by the termination codon TAG, and extending through 1 kbp of 3′ noncoding sequence of the NHX1 gene to a new SacI site at the 3′ end. The following primers were used in conjunction with C9410 as template: 5′-GACGTTCCAGATTACGCTGCTGAGTGCTAGCCGCGGGTAGACTTTAAAGTGTATGGTTTCC-3′ (sense) and 5′-GGCACGAGCTCGTCTTCATCCATGACGGAAG-3′ (antisense). The final PCR reaction used the PCR products, above, as primers with the plasmid pSM491 (gift of Susan Michaelis, Johns Hopkins University) containing the triple HA epitope as the template. The resulting 1.9-kbp product was digested with SpeI and SacI and cloned into pRin72 to give the full-length Nhx1::HA with the 1.9-kbp upstream sequence from the initiating codon ATG and the 1-kbp downstream sequence from the termination codon (pRin73). The Nhx1::GFP construct was created by digesting pEGFP-N3 (CLONTECH) with BamHI andNotI to release the 0.7-kilobase GFP and ligating to the same sites in pRin72. To complete the NHX1 open reading frame, a 1.3-kbp BamHI fragment from pRin73 was inserted in the correct orientation into this plasmid, creating the full-length fusion. kilobase pair polymerase chain reaction hemagglutinin green fluorescent protein prevacuolar compartment. Assays of protein, α-mannosidase activity, Kex2 activity, and azide-sensitive ATPase activity have been described in earlier publications (14Sorin A.G. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar) and in references therein. Differential centrifugation of yeast lysates and nonequilibrium fractionation of yeast lysates by sucrose gradient centrifugation was performed as described (14Sorin A.G. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 16Becherer K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. Cell. 1996; 7: 579-594Crossref PubMed Google Scholar). SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described previously (14Sorin A.G. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Antibodies were used as follows: mouse anti-HA antibody, 12CA5 (Boehringer Mannheim) at 1:5000, mouse anti-Vph1, monoclonal antibody 10D7-A7-B2 (Molecular Probes) at 1:5000, mouse anti-Dpm1, monoclonal antibody 5C5-A7 (Molecular Probes) at 1:2500, mouse anti-GFP (Molecular Probes) at 1:500, rabbit anti-Pma1 (gift of Carolyn Slayman, Yale University) at 1:1000, rabbit anti-Pep12 (gift of Robert Piper, University of Iowa) at 1:1250, rabbit anti-Kex2 (gift of Robert Fuller, University of Michigan),at 1:1000. Horseradish peroxidase-coupled goat anti-mouse (Boehringer Mannheim) and horseradish peroxidase-coupled donkey anti-rabbit (Amersham Pharmacia Biotech) were used at 1:1000. Cells (0.7–1.2A600 units/ml) were labeled with 53 μm FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienzyl)pyridinium dibromide), 13 nm DiOC6(3,3′-dihexyloxacarbocyanine iodide), and 40 nm MitoTracker Red CMXRos (all from Molecular Probes) using a variable labeling period (10–60 min) followed by chase in fresh medium (30–60 min). Confocal microscopy was performed by the Noran Oz Confocal Microscope System; single label controls for each fluorophore were captured under identical double label conditions to eliminate any fluorescence bleed-through. Targeted disruption of the S. cerevisiae NHX1 (YDR456w) gene leads to loss of sodium tolerance in acidic (Fig. 1 a) but not neutral or alkaline medium (12Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), consistent with the expected properties of a H+ driven Na+ transporter. The NHX1gene was recovered from a 40-kilobase pair genomic insert in cosmid C9410 (see “Experimental Procedures”), and the open reading frame was tagged at the C terminus with either a triple HA epitope or the GFP. Expression of the tagged constructs was directed from the endogenous NHX1 promoter in a Δnhx1 strain of yeast. Both tagged constructs appeared to be fully functional, effectively complementing the Na+-sensitive phenotype of the Δnhx1 mutant in the single copy (CEN) as well as multicopy (2μ) plasmid versions (Fig. 1 a). Like other members of the NHE family, yeast Nhx1 is predicted to be an integral membrane protein, with an N-terminal domain of 12 transmembrane helices, followed by a C-terminal cytoplasmic tail (12Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Differential centrifugation of yeast lysates results in a substantial enrichment of Nhx1::HA (molecular mass, 73.5 kDa) in low speed membrane pellets (Fig. 1 b); in the absence of further fractionation, the Nhx1 polypeptide characteristically migrates as multiple bands on SDS gels, indicative of post-translational modifications such as phosphorylation or glycosylation. Further evidence of the involvement of Nhx1 in halotolerance comes from salt induction of expression (Fig. 1 b). Our measurements of steady state intracellular 22Na levels indicated that enhanced sequestration of Na+ via Nhx1 correlated with salt-tolerant growth (12Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). By analogy with observations of vacuolar compartmentation of salt in halotolerant plants (17Serrano R. Int. Rev. Cytol. 1996; 165: 1-52Crossref PubMed Google Scholar, 18Barkla B.J. Apse M.P. Manolson M.F. Blumwald E. Symp. Soc. Exp. Biol. 1994; 48: 141-153PubMed Google Scholar), we hypothesized that Na+ transport by Nhx1 was likely to be coupled to the vacuolar H+ pump in an acidic compartment. Here, we show that HA-tagged Nhx1 cofractionates with markers for the vacuole, prevacuolar compartment, and the late Golgi compartment on sucrose density gradients (Fig.2 a), whereas it clearly fractionated away from markers representing the endoplasmic reticulum, plasma membrane, and mitochondria, pointing to a hitherto novel cellular location for a Na+/H+ exchanger. To distinguish between prevacuolar, vacuolar, and Golgi distributions, subcellular fractions from sequential centrifugation of yeast lysates were analyzed by gel electrophoresis (Fig. 2 b). Fractionation of Nhx1 closely followed that of the vacuolar marker, Vph1 of the vacuolar and that of prevacuolar but was clearly from the late Golgi To further the cellular location of this novel we used microscopy to the of Nhx1 in conjunction with the vacuolar FM 4-64 in cells (Fig. from Nhx1::GFP as fluorescent the vacuolar membrane, with a striking bipolar distribution. The and of the in consistent with the observed induction of Nhx1 expression The distinct location of the is of the prevacuolar compartment Mol. Biol. Cell. 1992; 3: PubMed Scopus Google Scholar, S.E. K. Emr S.D. Mol. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar), at the and endosomal for of final to the we show that the the of the prevacuolar compartment K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar) the that Nhx1 localizes to based on an of signals from the and Nhx1::GFP at levels from the was also for low levels of activity in of by other was not we show that fluorescence from Nhx1::GFP is distinct from that of two well characterized and MitoTracker Red CMXRos (Fig. as that have no orientation to the in Nhx1::GFP occurs as that are observed to abut the vacuolar membrane. with the results from subcellular fractionation (Fig. 2 a), we a localization for this exchanger. the of systematic have been identified in yeasts, worms, and provide a unique to the and of exchanger isoforms as well as provide to the of newly identified In of in we were to previously known of to the plasma membrane isoforms of Na+/H+ exchangers (NHE1–4), as well as a previously cluster of related We show that Nhx1 a completely novel cluster of exchanger from such as yeast, and humans a). 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In we show that there are consistent between members of the in sequence homology within transmembrane segments known to be important for amiloride and the of these we suggest that members of the cluster from plasma membrane isoforms. the of the C-terminal domain is in members of the cluster to the plasma membrane resulting in polypeptide versus residues. from by a or of members of the newly identified cluster are related to each these observations suggest a common intracellular, possibly endosomal location for these novel is evidence that plasma endosomes and transport vesicles at a prevacuolar compartment equivalent to late is to final to the to the vacuole may be in the as was observed for the from the plasma membrane M. J. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), or by of vesicle into or of this compartment, as in the family of vacuolar mutants J. Biol. 1995; PubMed Scopus Google Scholar). The is a than that be on density gradients from yeast Rieder S.E. Emr S.D. J. 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