From the Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada
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ABSTRACT |
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The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.
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INTRODUCTION |
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Electroneutral monovalent cation/proton antiporters or exchangers are important components of mitochondrial volume and Ca2+ homeostasis. In mammals, the latter is achieved by an integrated cycle of distinct Ca2+ uniport and Na+/H+ and Na+/Ca2+ antiport pathways located in the inner membrane (1-6). Matrix Ca2+ influences dehydrogenases of the tricarboxylic acid cycle (7, 8), F1F0-ATPase (9), and adenine nucleotide translocase (10, 11) and has been proposed as a mechanism for coordinating energy (ATP) production with cellular requirements (7, 8, 12). Although mitochondrial Ca2+ levels depend on Na+-dependent exchangers and the inner membrane Na+ and H+ gradients, the transporters have not been identified at the molecular level.
Functional studies suggest there are at least two distinct types of monovalent cation/proton exchangers in mammalian mitochondria. One of these preferentially mediates the exchange of matrix Na+ for intermembrane H+ generated by respiration (i.e. a Na+-selective Na+/H+ exchanger (NHE)1) (1, 13). This mechanism also transports Li+ (and possibly NH4+) at a much lower rate and is inhibited by benzamil derivatives of amiloride (14-17). It is constitutively active in respiring mitochondria and is primarily responsible for establishing the [Na+] gradient ([Na+]i < [Na+]o) that allows Na+-dependent extrusion of matrix Ca2+ (18). The other monovalent cation/H+ exchanger is latent, transports all alkali cations (i.e. Li+, Na+, K+, Rb+, and Cs+) at similar rates, and is antagonized by quinine, dicyclohexylcarbodiimide, and propranolol (19-21). Since K+ is the predominant intracellular alkali cation, it is usually referred to as a K+/H+ exchanger. In isolated mitochondria, it is active only upon depletion of matrix Mg2+ and simultaneous elevation of matrix pH or upon hypotonic swelling (19, 21, 22). It is postulated to play a role in organellar volume homeostasis (23). Mitochondria from Saccharomyces cerevisiae possess a similar nonselective K+/H+ exchanger, although the yeast transporter can be activated without depletion of matrix Mg2+ (24). However, functional studies of yeast mitochondria have not revealed any Na+-selective NHE activity (25).
In addition to their role in mitochondria, mammalian NHEs participate in a wide array of other essential cellular processes, including control of intracellular pH (pHi), maintenance of cellular volume, and reabsorption of Na+ across renal, intestinal, and other epithelia (26, 27). Activation of NHE activity also facilitates growth factor-induced proliferation of certain cell types (27) and is associated with events leading to apoptosis (28-30). To date, five NHE isoforms (NHE1 to NHE5) have been identified that exhibit considerable differences in their primary structures (34-60% identity), tissue distribution, membrane localization, biochemical and pharmacological properties, and responsiveness to various stimuli (31, 32). While characterization of some of these isoforms is incomplete, the existing data do not support a direct role for any of them in mitochondrial function.
To identify mitochondrial type NHEs, we have focused our attention on S. cerevisiae because the sequence of the entire genome is known and because of the ease with which this organism can be manipulated genetically and analyzed functionally. In addition, yeast are facultative aerobes that can proliferate in the absence of aerobic respiration, which permits in vivo studies of mutations that disrupt mitochondrial function. To date, a S. cerevisiae gene (NHA1) conferring tolerance to increasing concentrations of external NaCl has been isolated (33), and its protein product was found to be homologous (40% amino acid identity) to the cell surface Na+/H+ antiporter Sod2 of Schizosaccharomyces pombe, which is essential for Na+ extrusion (34). Further examination of the S. cerevisiae genome has revealed additional anonymous open reading frames (YDR456w, YJL094c, YJR106w, YAL022c) whose encoded protein sequences show similarity to Na+/H+ exchangers. YDR456w is of particular interest, since PSORT analysis (35) predicts that its protein product would be situated in the mitochondrial inner membrane due to potential targeting signals at its N terminus. To test this hypothesis, we isolated the YDR456w gene (renamed NHA2) and show that its protein product is localized to yeast mitochondria. We also characterized its function in isolated mitochondria and demonstrated its physiological role by comparing wild-type yeast with an isogenic strain lacking this gene. Yeast NHA2 also has strong sequence similarity to a partial cDNA (KIAA0267) that was recently cloned from a human bone marrow cell line.2 We report the complete primary structure and tissue distribution of this novel human isoform (renamed NHE6) and show that it is also localized exclusively in mitochondria.
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EXPERIMENTAL PROCEDURES |
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Materials--
Carrier-free 22NaCl (range of
specific activity, 300-900 mCi/mg; concentration, ~5 mCi/ml) was
obtained from NEN Life Science Products (Mandel Scientific, Guelph,
Ontario, Canada). Benzamil, quinine, rotenone, and phenylmethylsulfonyl
fluoride were purchased from Sigma. -Minimal essential medium, fetal
bovine serum, penicillin/streptomycin, and trypsin-EDTA were purchased
from Life Technologies (Burlington, Ontario). Cell culture dishes and
flasks were purchased from Becton Dickinson and/or Corning
(Montréal, Québec, Canada). All other chemicals and
reagents used in these experiments were purchased from BDH Inc. (St.
Laurent, Québec, Canada) or Fisher and were of the highest grade
available.
Strains and Media--
The S. cerevisiae strains used
in this study are SEY6210 (MAT, leu2-2, ura3-52,
his3-
200, lys2-801, trp1-
901; kindly provided by Dr. Marc
Lussier, McGill University) (36) and L4244 (MAT
, leu2-
1,
ura3-52, his3-
200) (kindly provided by Dr. Gerald R. Fink,
Whitehead Institute, Cambridge, MA). A rich medium (YPD) containing 1%
Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose or a minimal
medium (SD) containing 0.67% yeast nitrogen base and 1% dextrose
supplemented with required amino acids was used for standard growth of
S. cerevisiae. To analyze mitochondrial function, 2% of
galactose, glycerol, succinate, or lactate was substituted as the
carbon source.
Measurement of Yeast Survival in the Stationary Phase--
Wild
type and the corresponding nha2 strains were inoculated
into 10 ml of YPD medium and grown at 30 °C in a shaking platform incubator. Following incubation for 2, 10, and 20 days, optical densities of the cultures were determined by absorbance at 600 nm.
Aliquots of equivalent optical densities were plated onto YPD plates
and cultured for a further 2 days.
Molecular Cloning-- The anonymous open reading frame YDR456w in S. cerevisiae corresponding to gene D9461.40 (renamed NHA2) was cloned by polymerase chain reaction (PCR) based on the known sequence (GenBankTM accession number U33007). The sequence identity and fidelity was confirmed by DNA sequencing.
A partial human cDNA (KIAA0267) showing homology to members of the NHE gene family was generously provided by Dr. T. Nagase (Kazusa DNA Research Institute, Kisarazu, Japan). To obtain the full-length sequence, we extended the partial cDNA sequence by 5'-rapid amplification of its cDNA end (5'-RACE) (37) using Marathon-ReadyTM cDNAs from human kidney (CLONTECH, Palo Alto, CA) as a template. For the initial PCR, the 5'-oligonucleotide primer was AP1, which corresponded to a sequence within the 5'-adaptor (CLONTECH), and the 3'-primer (5'-GGCTTGCTTCTCGGACACG-3') was derived from the 5'-sequence of KIAA0267. Five µl of the initial 25-µl PCR product was reamplified with a nested 5'-adaptor primer AP2 (CLONTECH) and a nested 3'-primer (5'-GCTTCTCGGACACGATCTCCTCGTCC-3'). The PCR products were resolved by electrophoresis on agarose gels, isolated, subcloned into the pGEM-T vector (Promega, Madison, WI), and analyzed by DNA sequencing. Then, a NotI-NcoI fragment was isolated from one of the 5'-RACE products containing the missing sequence and ligated to the partial cDNA to obtain the full-length sequence.Mammalian Cell Culture--
HeLa cells were maintained in
complete -minimal essential medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 mm of NaHCO3 (pH 7.4) and incubated in a humidified
atmosphere of 95% air and 5% CO2 at 37 °C.
Northern Blotting-- A human poly(A+) RNA blot was purchased from CLONTECH and hybridized with a 0.6-kilobase pair ClaI-EcoRI fragment isolated from the 3'-end of the cDNA that exhibited little sequence identity to the other known mammalian NHE isoforms. The probe was radiolabeled with [32P]dCTP by the random primer method (Pharmacia Biotech Inc.) and hybridized at 68 °C for 1 h in the Express HybTM solution (CLONTECH). The blot was washed twice in 2× SSC, 0.05% SDS at room temperature for 30 min each followed by a high stringency wash in 0.1× SSC, 0.1% SDS at 50 °C for 40 min. The radioactive signals were analyzed using a PhosphorImager (Molecular Dynamics).
Gene Disruption in Saccharomyces cerevisiae-- Creation of null alleles in yeast was accomplished using the PCR-based gene disruption technique (38, 39). This was accomplished using the pBM2983 plasmid carrying a gene replacement cassette, which encompasses a promoterless green fluorescent protein (GFP) open reading frame from the jellyfish Aequorea victoria and a tandemly located HIS3 marker with its own promoter (kindly provided by Dr. J. H. Hegemann, Justus-Liebig-Universität Giessen, Germany) (39). GFP emits green fluorescence (sharp peak at ~508 nm) when excited between 450 and 490 nm and can be followed in both living and formaldehyde-fixed cells. The presence of GFP in the replacement cassette is useful for examining the regulation of the endogenous NHA2 promoter, although this was not undertaken in the current study. The replacement gene was amplified by PCR using the following primers. Primer 1 (5'-oligonucleotide) contains 45 nucleotides immediately 5' of the ATG start codon of NHA2 followed by 19 nucleotides of the GFP coding region beginning at the ATG start codon (5'-TCAGAGGATAATCTTTTATCGCTGTCAGTACATCAACTATGAAAAATGAGTAAAGGAGAA-3'); primer 2 (3'-oligonucleotide) contains 45 nucleotides immediately 3' of the stop codon of NHA2 followed by 19 nucleotides of the HIS3 coding region beginning 154 bp downstream of the HIS3 stop codon (5'-TTAATATATTTATATTAGAAACAAGGAAACCATACACTTTAAAGTGCGCGCCTCGTTCAG-3'). The PCR fragment was transformed into the SEY6210 haploid yeast strain by the lithium acetate/single-stranded DNA/polyethylene glycol method (40) and grown on histidine-deficient medium. Homologous recombinants were selected by using the HIS3 auxotrophic marker and expression of GFP (under the control of the NHA2 promoter). Deletion of NHA2 was confirmed by PCR analysis of genomic DNA using specific primers to an upstream region of the NHA2 promoter and an internal sequence of GFP.
Construction of Na+/H+ Exchanger-Green Fluorescent Protein Chimeras-- The PCR fragment corresponding to the entire open reading frame of S. cerevisiae NHA2, but excluding the termination codon, was digested with XbaI and ClaI and cloned into the corresponding site in the yeast expression vector pGFP-C-FUS (kindly provided by Dr. Hegemann) (39). This resulted in an NHA2-GFP fusion construct under the control of the methionine-repressible MET25 promoter (pGFP-C-FUS-NHA2). To construct a human NHE6-GFP chimera, an XbaI site was introduced by PCR at the C-terminal end of the NHE6 open reading frame just before the stop codon. A NotI-XbaI fragment containing the entire NHE6 coding sequence was then fused in frame with the XbaI-EcoRI fragment of cDNA encoding a modified GFP exhibiting enhanced fluorescence (GFP-cycle 3) (41), and then subcloned into the mammalian expression vector pIRES-neo (CLONTECH).
Subcellular Localization-- The pGFP-C-FUS control plasmid and pGFP-C-FUS-NHA2 fusion construct were transformed into SEY6210 haploid strain and URA+ colonies were selected on SD plates supplemented with Leu, His, Trp, and Lys. Transformed cells were inoculated into SD medium devoid of uracil and methionine to maintain selection and to induce high levels of expression from the MET25 promoter, respectively. To stain mitochondria in living cells, exponentially growing cells were incubated in SD medium plus 2.5 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) for 30 min at 30 °C, followed by fixation by incubation for 30 min in SD medium containing 3.7% formaldehyde. Under these conditions, mitochondrial DNA is preferentially stained compared with nuclear DNA (42). The transformed cells were examined by fluorescence microscopy (Nikon) with the appropriate filter sets (filter set UV-1A for DAPI: excitation 365 ± 10 nm, emission 400 nm; filter set B-2A for GFP: excitation 450-490 nm, emission 520 nm).
HeLa cells were transiently transfected with the human NHE6-GFP fusion construct by the calcium phosphate method (43). Following transfection, cells were kept for 24 h at 37 °C and then incubated at a reduced temperature (30 °C) for another 36 h to enhance the fluorescence signal of GFP, as described previously (44). Mitochondria were selectively stained by incubating the cells with 25 nM of the MitoTracker Red CM-H2XRosTM dye (Molecular Probes, Eugene, OR) for 20 min at 37 °C. This dye is not fluorescent until it enters metabolically active cells, where it is oxidized to the fluorescent probe and sequestered into mitochondria. The results were analyzed by confocal laser microscopy using a Zeiss LSM 410 inverted microscope. The MitoTracker Red CM-H2XRosTM dye was excited with a helium/neon (543-nm) laser and was imaged on a photomultiplier after passage through FT560 and LP590 filter sets. The NHE6-GFP was imaged by exciting the sample with a 488-nm line from an argon/krypton laser, and the resulting fluorescence was collected on a photomultiplier after passage through FT510 and BP515-540 filter sets. All images were line averaged and printed on a Kodak XLS8300 high resolution (300 dots per inch) printer. Optical sections were taken using a × 63, 1.4 NA objective (optical thickness, ~1.0 µm).Measurement of 22Na+ Influx into Isolated
Mitochondria--
Mitochondria from S. cerevisiae wild-type
and nha2 strains, which were grown on 2%
glycerol-containing medium, were isolated by differential
centrifugation as described by Yaffe (45). The mitochondrial pellet was
resuspended in 0.25 M sucrose, 2 mM Tris-HCl (pH 7.4) in a concentration of 50 µl/g of original cells. NHE activity was assessed by measuring 22Na+ fluxes
as described previously (14). Briefly, mitochondria (75 µg) were
treated with 4 µg/ml rotenone in 300 µl of buffer containing 100 mM mannitol, 45 mM sodium acetate, 20 mM TES (pH 7.2), and 1 mM EGTA for 6 min at
room temperature. The mitochondrial suspension was incubated with 1.5 µCi of 22Na+ for 15 s, immediately
diluted in 5 ml of ice-cold stop solution (0.1 M LiCl),
filtered through a 0.3-µm Millipore filter (Millipore Corp., Bedford,
MA), and washed three times with 5 ml of ice-cold stop solution. Each
experiment was replicated 3-5 times from the same mitochondrial
preparation.
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RESULTS |
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Subcellular Localization of S. cerevisiae Nha2--
Analysis of
the primary structure of S. cerevisiae Nha2 using PSORT
(35), which estimates the subcellular localization of proteins in
eukaryotic cells based on known targeting motifs, predicted that it
would be localized in the mitochondrial inner membrane due to targeting
signals at its N terminus. These signals are highly degenerate residues
(20-30) capable of forming a positively charged amphiphilic helix,
which direct the protein to the mitochondrial matrix, followed by one
or more hydrophobic -helical segments that arrest the protein in the
membrane (46). To test the hypothesis that Nha2 is targeted to
mitochondria, we amplified the NHA2 gene by PCR from
S. cerevisiae genomic DNA and inserted it into the green
fluorescent protein fusion vector pGFP-C-FUS (39) in the proper open
reading frame to create an NHA2-GFP fusion gene (GFP at the
C terminus of Nha2) under the control of the methionine-regulated MET25
promoter. The sequence of NHA2 was verified to ensure that mutations were not introduced during gene amplification. This approach
has been used successfully for studying the expression and subcellular
localization of other genes in budding yeast (39). The
pGFP-C-FUS-NHA2 fusion plasmid and the pGFP-C-FUS control plasmid were transformed into the yeast strain SEY6210 and maintained in medium lacking uracil (to select for transfectants) and methionine (to induce high levels of expression from the MET25 promoter). GFP
fluorescence was monitored in formaldehyde-fixed yeast cells using
fluorescence microscopy. Mitochondria were detected using the
fluorescent DNA-binding dye, DAPI, under conditions that stain yeast
mitochondrial DNA more intensely than nuclear DNA (42). As shown in
Fig. 1A, DAPI staining was
observed at discrete sites along the periphery of a representative
cell, which are presumed to be mitochondria, and these co-localized
with the fluorescence signal of the Nha2-GFP fusion protein (Fig.
1B). By contrast, expression of unfused GFP, which lacks
membrane targeting motifs, showed a more diffuse green fluorescence
throughout the cytoplasm (Fig. 1C). These data are
consistent with the localization of Nha2-GFP to mitochondria.
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Functional Characterization of S. cerevisiae Nha2 Protein--
To
define the function of yeast Nha2, the entire open reading frame of
NHA2 was replaced with a cassette containing the
HIS3 auxotrophic marker. Gene disruptants were selected by
their ability to grow on histidine-deficient medium, and deletion of
NHA2 was confirmed by PCR analysis (data not shown). As
illustrated in Fig. 2, mitochondria
isolated from the wild-type strain displayed measurable acid-induced
22Na+ influx that was inhibited by 100 µM benzamil, an amiloride analogue that preferentially
antagonizes mitochondrial NHE activity (14-17) compared with plasma
membrane NHEs (47, 48). 22Na+ influx was also
inhibited by Li+ (100 mM), which antagonized
Na+ transport (data not shown). By comparison,
benzamil-inhibitable 22Na+ influx was absent in
the nha2 mitochondria. These data support the notion that
Nha2 is a functional Na+/H+ exchanger in
yeast mitochondria.
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Similarity of Nha2 with Other Na+/H+ Exchangers-- An extensive search (50) of sequences in the GenBankTM data base identified putative NHE homologues in Caenorhabditis elegans (F57C7.2) and Homo sapiens (KIAA0267) with strong amino acid identity (27 and 30%, respectively) to S. cerevisiae Nha2. C. elegans F57C7.2 and H. sapiens KIAA0267 exhibited even higher identity (34%) to each other. By comparison, H. sapiens KIAA0267 showed lower amino acid identity (~20-24%) to other mammalian NHE isoforms (NHE1-NHE5). The closer relatedness of C. elegans F57C7.2 and H. sapiens KIAA0267 to S. cerevisiae Nha2 suggested that they may fulfill a common function.
Further examination of the human KIAA0267 cDNA indicated that the 5'-region seemingly lacked nucleotide sequence containing the initiation methionine codon, although a potential in frame AUG start site is present 67 nucleotides from the extreme 5'-end. To determine if other possible start codons exist upstream, we extended the sequence by 5'-RACE (37) using nested oligonucleotide primers and a single-stranded cDNA library generated from human kidney mRNA as a template. One of the generated PCR products (Fig. 5A) contained 175 bp of overlapping sequence with the 5'-region of KIAA0267 and an additional 44 nucleotides, which encoded three amino acids starting from an AUG in frame with the putative open reading frame. This AUG codon is preceded by an in frame TAG stop codon at nucleotide position
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Tissue Distribution of Human NHE6-- If NHE6 is a mitochondrial protein, one would expect it to be broadly distributed among mammalian tissues. To test this hypothesis, a Northern blot containing poly(A+) RNA isolated from several adult human tissues was hybridized at high stringency with an isoform-specific, 32P-radiolabeled NHE6 probe (0.6 kilobase pair) obtained from the 3'-end of the cDNA. As shown in Fig. 6, NHE6 mRNA was detected as a single band of ~5.5 kilobase pairs in all tissues examined, with highest abundance in brain and skeletal muscle, followed by heart and lower amounts in the other tissues. This expression pattern is distinct from that of the other NHE isoforms (32, 55) and is generally consistent with the relative abundance of mitochondria in these tissues (56).
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Subcellular Localization of Human NHE6-- To determine if NHE6 is targeted to mitochondria as predicted, dual labeling experiments were carried out in HeLa cells using a mitochondrion-specific marker, MitoTracker Red CM-H2XRosTM, and transient expression of a chimeric protein composed of NHE6 fused at its C terminus to GFP. As demonstrated in a binucleate HeLa cell (Fig. 7), the red fluorescence from the MitoTracker dye (Fig. 7A) was found primarily in clusters of punctate extranuclear sites, consistent with mitochondrial localization. The green signal emitted by the NHE6-GFP protein (Fig. 7B), although somewhat more diffuse, was also punctate and accumulated in identical subregions of the cell. Co-localization of signals from the MitoTracker Red dye and NHE6-GFP is indicated in yellow when the red and green scans are overlaid (Fig. 7C). This pattern represents the staining seen in all experiments. These data suggest that NHE6 is most likely targeted to mitochondria, although other possibilities, such as artifactual aggregation of the fusion protein, cannot be excluded.
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DISCUSSION |
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BLAST analysis of the deduced protein sequences from three uncharacterized genes, one from S. cerevisiae (i.e. YDR456w or NHA2), one from humans (i.e. KIAA0267 or NHE6), and the other from C. elegans (i.e. F57C7.2), identifies them as homologues of the Na+/H+ exchanger gene family. Moreover, these three sequences exhibit higher amino acid identity to each other (27-34%) than they do for other NHE isoforms within their respective species, suggesting that they may share a common function. In the case of S. cerevisiae Nha2 and human NHE6, PSORT analysis predicts that their extreme N-terminal regions contain targeting signals that direct proteins to the mitochondrial inner membrane. Accordingly, we tested this prediction by fluorescence microscopy and found that they were indeed localized to mitochondria. Furthermore, we found a wide distribution and high expression of human NHE6 mRNA in tissues with abundant mitochondria (i.e. brain, skeletal muscle, heart), consistent with a role for this isoform in mitochondrial function. It is noteworthy that pathological changes resulting from mitochondrial dysfunction are also manifested predominantly in these same tissues (56). The absence of a putative mitochondrial targeting motif in the F57C7.2 protein of C. elegans may indicate a different cellular role for this NHE homologue or simply reflect incomplete sequence at its N terminus.
While the functional properties of the mammalian Na+-selective NHE are well described (14-17), there is little information concerning its structural features. Using subcellular fractionation and gel electrophoresis, Garlid et al. (13) identified a major 59-kDa protein in beef heart submitochondrial particles that was associated with Na+-selective NHE activity. This value approximates the molecular mass calculated for human NHE6 (~74 kDa), particularly since hydrophobic membrane proteins tend to migrate faster in polyacrylamide gels relative to other proteins of the same mass, which results in an underestimation of their true size. Proteolytic removal of the matrix targeting signal upon insertion into the inner membrane may also contribute to an observed smaller mass of the mature protein, as has been reported for other similarly targeted mitochondrial proteins (46). However, whether the 59-kDa protein and NHE6 are the same protein remains uncertain. The yeast Nha2 also has a molecular mass in this range (~70 kDa). By contrast, the mammalian nonselective K+/H+ (Na+/H+) exchanger has been tentatively identified as an 82-kDa protein based on radioisotope labeling of rat liver mitoplast extracts with [14C]dicyclohexylcarbodiimide, which irreversibly inhibits the transporter (20).
Predictions of secondary structure indicate that NHE6 and Nha2 have the
same membrane topology as other NHEs, with 12 -helical hydrophobic
membrane-spanning segments in the N terminus followed by a hydrophilic
region at the C terminus. The N terminus contains a single putative
N-linked glycosylation site (Asn369-Leu-Ser) in
the predicted extracellular loop between membrane-spanning segments 9 and 10, which is highly conserved in eukaryotic NHEs. However, this
site does not appear to be glycosylated in the NHE isoforms
(i.e. NHE1, NHE2, and NHE3) that have been examined to date
(57-59); therefore, its significance is obscure. The cytoplasmic region of NHE6 contains putative recognition sites (60) for phosphorylation by cyclic nucleotide-dependent protein
kinases (i.e. 524RRTT, 570RCLT, and
643RGT) and casein kinase II (i.e.
512SDQE, 527TKAE, 601TYGD, and
624SSED). Whether protein kinases regulate NHE6 activity is
unknown; nevertheless, this remains a distinct possibility, since
cAMP-dependent protein kinase I and II (61) and casein
kinase II (62) are located at the inner membrane/matrix space of
mitochondria. By comparison, plasma membrane NHEs (e.g.
NHE1, NHE2, and NHE3) are highly regulated phosphoproteins that contain
multiple consensus phosphorylation sites recognized by various protein
kinases in their C termini, some of which are known to be
phosphorylated (63).
The chromosomal copy of NHA2 present in SEY6210 haploid cells was deleted by insertion of the HIS3 auxotrophic marker into its open reading frame. Comparison of 22Na+ uptake into the acidified matrix of intact mitochondria isolated from the wild-type and the isogenic NHA2 gene-disrupted strains indicated that Nha2 is capable of mediating Na+/H+ exchange that is inhibited by benzamil and Li+. Benzamil has been shown to preferentially inhibit the mammalian mitochondrial Na+-selective NHE at the concentration (0.1 mM) used in this study (16), whereas it is a poor inhibitor of plasma membrane NHEs. This suggests that yeast Nha2 has pharmacological properties similar to those of its mammalian homologue.
These pharmacological data seem to contradict an earlier study that reported that mitochondria from S. cerevisiae have a nonselective K+/H+ (Na+/H+) exchanger but lack a discernible Na+-selective NHE (25). However, the low signal-to-noise ratio for measuring Nha2 activity may have caused it to go undetected in the earlier study, because we found that it was difficult to measure in our mitochondrial preparations. Indeed, benzamil-inhibitable Nha2 activity (1 nmol/min/mg protein) was only detected in three of five of our mitochondrial preparations. Our preliminary attempts to determine if Nha2 activity is sensitive to quinine, an inhibitor of the nonselective cation/H+ exchanger, were inconclusive. By comparison, Na+-selective NHE activity in heart mitochondria is about 100-fold higher (110-160 nmol/min/mg protein), which facilitates pharmacological and kinetic measurements (2, 16). Attempts to measure matrix pH (as an assay of Na+/H+ exchange activity) using the membrane-permeant dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester were unsuccessful. Others have also reported that yeast mitochondria do not accumulate fluorescent indicators when present as membrane-permeant esters (64), perhaps because they lack the esterases required for cleavage of the acetoxymethylester group. We are currently manipulating the assay conditions and improving the mitochondrial preparation in an attempt to increase the specific rates of transport by Nha2. At present, we cannot determine if Nha2 functions as a Na+-selective NHE or as a nonselective cation/H+ exchanger. It is worth noting that, aside from Nha1 (located at the cell surface) (33) and Nha2, S. cerevisiae contains three additional anonymous open reading frames (YJL094c, YJR106w, and YAL022c) that share weak similarity to Na+/H+ exchangers. Of these, YJL094c also shows closest homology to a glutathione-regulated K+ efflux transporter (KefC) from Escherichia coli (65), although it does not appear to contain a putative mitochondrial targeting signal at its N terminus. Determination of their transport properties and cellular location may clarify the role of Nha2.
Additional evidence supporting a role for Nha2 in organellar Na+ transport was provided by a very recent study that showed that deletion of NHA2 in S. cerevisiae (referred to as NHX1 in this study) decreases the Na+ tolerance of a yeast strain expressing a mutant plasma membrane H+-ATPase (66). It was speculated that Nha2 regulated the sequestration of Na+ into an intracellular acidic compartment, possibly vacuoles, although localization to mitochondria was not considered.
In the present study, initial characterization showed that yeast cell
growth on fermentable carbon sources (i.e. glucose) was
unaffected by the loss of NHA2 (data not shown).
Under these growth conditions, yeast derive their energy primarily from
glycolysis rather than respiration. The tolerance of nha2
cells to 100 mM concentrations of external cations
(i.e. Li+, Na+, K+, or
Ca2+) was also unaltered when grown on regular YPD medium
(data not shown). However, further analysis of the
nha2
cells revealed that mitochondrial function was compromised. The growth
of
nha2 cells on nonfermentable carbon sources was
retarded when compared with the parental strain, suggesting that energy
production from aerobic metabolism was reduced.
To further examine mitochondrial function, we investigated the ability
of these yeast strains to survive in the nonproliferative (i.e. stationary) phase of the cell cycle upon nutrient
depletion (49). When exponentially growing cells exhaust their supply of glucose, they adapt to respiratory metabolism using nonfermentable carbon sources generated by glycolysis. At this stage, the cells resume
growth by using the energy from oxidative phosphorylation, but at a
greatly reduced rate. The cells eventually enter the stationary phase
upon carbon starvation (around day 6 of growth). During this
transition, wild-type yeasts undergo numerous biological adaptations
that allow them to survive over several weeks without added nutrients
and quickly resume growth when nutrients become available. As
demonstrated in this study, both wild-type SEY6210 and isogenic
nha2 cells survived equally well in glucose-rich medium
during the early stages (day 2) of cell growth, but, when sampled
during the stationary phase (day 10), the survival of the
nha2 cells was dramatically reduced, with the vast
majority of the remaining cells exhibiting a petite
phenotype. By contrast, the wild-type cells showed no detectable loss
of viability.
Interestingly, a few nha2 cells did retain wild-type
growth (large colony size) in the absence of glucose, suggesting that the defect caused by the loss of Nha2 activity may be either weakly penetrating or that disruptants can spontaneously develop a suppressor activity that reduces severity of the gene loss. These explanations would also account for the modest reduction in growth of
nha2 on nonfermentable carbon sources. Indeed, we have
recently observed a variation in the strength of the null-mutant
phenotype with different disruptions. Using another yeast strain,
L4244, more severe effects on mitochondrial function were obtained
following homologous disruption of the NHA2 gene. Unlike the
parental strain, the L4244
nha2 cells showed retarded
growth on regular YPD medium and no growth on nonfermentable carbon
sources (data not shown). These data suggest that the SEY6210 strain
contains a secondary mechanism, which partly complements the effects of
deleting the NHA2 gene and which is absent in the L4244
strain. One possibility could be the activation of another Nha2-related
antiporter, although other explanations are equally plausible. We are
currently attempting to characterize this compensatory mechanism, which
would help clarify the role of Nha2 in mitochondrial function.
The mechanism whereby disruption of NHA2 leads to a
deterioration of yeast viability is unknown. Unlike mammalian
mitochondria, yeast mitochondria apparently lack a high affinity
Ca2+ uniporter (67, 68). Na+/Ca2+
antiporter activity also remains to be demonstrated in yeast mitochondria. Hence, the main physiological role of Nha2 in
yeast mitochondria might be to control organellar volume rather than Ca2+ homeostasis. If so, then respiring mitochondria of
nha2 cells may swell excessively because of their
inability to extrude matrix Na+ (and possibly
K+) that enters via various leak pathways (driven by the
negative membrane potential) (68). In addition, a reduction in
H+ influx through Nha2 may increase matrix pH.
Both mitochondrial swelling and matrix alkalinization promote the
opening of the yeast mitochondrial permeability transition pore, which
mediates the equilibration of solutes (
1.5 kDa) across the inner
membrane (64). Chronic opening of the mitochondrial permeability
transition pore disrupts oxidative ATP production and could result in
irreversible mitochondrial damage and cell death. Studies are currently
under way to test this hypothesis.
In summary, we have characterized a novel Na+/H+ exchanger isoform (Nha2) found in yeast, which is targeted to mitochondria, mediates Na+/H+ exchange, and is important for mitochondrial function. Furthermore, we have also demonstrated that a related gene product (which we named NHE6) in humans is also localized in mitochondria. We propose that both Nha2 and NHE6 are important for the regulation of intramitochondrial Na+ and H+ levels. The yeast system should facilitate further studies on the mechanisms and functional importance of this transporter in eukaryotic cells.
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ACKNOWLEDGEMENT |
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We thank Dr. J. Hanrahan (McGill University) for useful discussions and for critically reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada.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.
Supported by a research scholarship from the Fonds de la Recherche
en Santé du Quebec. To whom correspondence should be addressed: Dept. of Physiology, McGill University, McIntyre Medical Science Bldg.,
3655 Drummond St., Montreal, Québec H3G 1Y6, Canada. Tel.: 514-398-8335; Fax: 514-398-7452; E-mail:
orlowski{at}physio.mcgill.ca.
1 The abbreviations used are: NHE, Na+/H+ exchanger; PCR, polymerase chain reaction; 5'-RACE, 5'-rapid amplification of cDNA end; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; GFP, green fluorescent protein; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid.
2 T. Nagase, N. Seki, K. Ishikawa, O. Ohara, and N. Nomura, GenBankTM accession number D87743.
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