The LETM1 / YOL027 Gene Family Encodes a Factor of the Mitochondrial K (cid:1) Homeostasis with a Potential Role in the Wolf-Hirschhorn Syndrome*

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene ( yol027 (cid:2) mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1 / Yol027 proteins from yeast to man. Mutant yol027 (cid:2) mitochondria have increased cation contents, particularly K (cid:1) and low-membrane-poten-tial (cid:2)(cid:3) . They are massively swollen in situ and refrac-tory to potassium acetate-induced swelling in vitro , which is indicative of a defect in K (cid:1) /H (cid:1) exchange activity. Thus, YOL027 / LETM1 are the first genes shown to encode factors involved in both K (cid:1) 5 (cid:5) primer carrying a natural SacI site and a 3 (cid:5) primer that introduced a PstI site upstream of the stop codon. The SacI-PstI fragment of this product was inserted into YCp33-HA vector, resulting in plasmid YCp- YOL027 -HA. YPR125 coding sequence (1356 nucleotides from 1 to (cid:4) 1356) was PCR-amplified from the same yeast strain, introducing recognition sites for EcoRI and SalI, followed by insertion of this fragment into the EcoRI and SalI sites of the centromeric vector pUG35. The resulting plasmid pUG35- YPR125 -GFP expressed the YPR125 gene under the MET25 promoter and in-frame with the GFP coding sequence following at its 3 (cid:5) end. The YEp- YPR125 construct (YEp-MW7) had been cloned by Waldherr et al (10). To clone the human LETM1 cDNA, SuperScript II reverse tran-scriptase (Invitrogen) and an oligo(dT) primer were used for first-strand synthesis on poly(A)-enriched template RNA isolated from a PA-1 teratocarcinoma cell line. PCR fragments covering the entire cod- ing sequences (from nucleotide 1 to (cid:4) 2590) were amplified, and the purified PCR product was cloned to the pGEM-T vector (Promega), and a C-terminal HA-tag was added by PCR. The PCR product was digested with XhoI and HindIII and cloned into the corresponding sites of the yeast expression vector, pVT103-U. An XhoI-EcoRI PCR fragment was cloned into the pEGFP-N1 mammalian expression vector, thus creating an in-frame fusion with the enhanced GFP (EGFP) sequence carried on the vector. Gene Deletion— Complete disruptions of YOL027 , MRS2 , and MRS7 were performed according to the one-step replacement protocol de- scribed in Ref. 16 in the diploid yeast strain W303 and the haploid DBY747. Disruption of YOL027 resulted

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027⌬ mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027⌬ mitochondria have increased cation contents, particularly K ؉ and low-membrane-potential ⌬⌿. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K ؉ /H ؉ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K ؉ homeostasis and organelle volume control.
Respiring mitochondria maintain a membrane potential (⌬⌿) 1 of Ϫ150 to Ϫ180 mV (⌬⌿, inside negative). This high ⌬⌿ constitutes a large driving force for the electrophoretic influx of cations, either through specific channels or by diffusion through the membrane. Several cation channels have been characterized physiologically (reviewed in Refs. 1 and 2), and recently, a single one has been identified molecularly (3). These transport systems seem to have intrinsic control mechanisms which ensure that the matrix cation concentrations stay within physiological ranges, far below chemical equilibrium.
Diffusive permeability of the inner mitochondrial membrane to ions is generally low but physiologically significant, as it lowers the pH gradient and membrane potential. Moreover, if not counteracted by extrusion, steadily increasing concentrations of matrix cations (and of compensating anions) will lead to an imbalance of osmotic pressure across the inner mitochondrial membrane. As a consequence, water will pass through the membrane, causing excessive swelling and eventual rupture of the organelle (1,2,4).
As first proposed by P. Mitchell (5), mitochondria have carrier systems allowing the electroneutral exchange of cations against H ϩ (and anions against OH Ϫ ). These exchangers counteract the ⌬⌿-driven cation leakage of the membrane and also cation imbalances due to changes in mitochondrial physiology. Mitochondrial cation distribution is, therefore, a steady state, in which the accumulation ratio is modulated by the relative rates of cation influx and efflux by means of separate pathways.
Many physiological studies have been devoted to cation/H ϩ exchange systems (reviewed in Ref. 1). With respect to the most abundant cations in cells and mitochondria, K ϩ (150 mM) and Na ϩ (5 mM), researchers agree on the existence of two separate antiporters in mammalian cells, a selective Na ϩ /H ϩ exchanger, and an unselective K ϩ /H ϩ exchanger transporting virtually all alkali ions. Given the particularly high concentration of K ϩ in cells and mitochondria, the unselective exchanger is referred to most commonly as the K ϩ /H ϩ exchanger (reviewed in Ref. 1). This exchanger has pronounced sensitivity to matrix [Mg 2ϩ ] (K i of 0.3-0.4 mM in mammalian mitochondria), timolol, and quinine. Proteins of apparent molecular masses of 82 and 59 kDa constitute the unselective mitochondrial K ϩ /H ϩ exchanger and the selective Na ϩ /H ϩ exchanger, respectively (6,7). Attempts to identify the gene encoding the K ϩ /H ϩ have not been successful yet (2), and a report on the identification of the yeast NHE2 and its mammalian homolog NHE6 as encoding the mitochondrial Na ϩ /H ϩ exchanger (8) have been questioned recently (9).
In the course of characterizing a set of yeast genes potentially encoding mitochondrial cation transport proteins (10) we focused on the yeast genes MRS7 and YOL027, as well as their human homolog LETM1 (leucine zipper/EF-hand-containing trans-membrane domain; Ref. 11), which are representatives of a novel eukaryotic gene family with hitherto unknown function. Hemizygous deletion of a region on human chromosome 4 (4p16.3), including LETM1 and several other genes, causes the Wolf-Hirschhorn syndrome. Recent data reveal that the full Wolf-Hirschhorn syndrome phenotype, including neuromuscular features, such as seizures correlates with the deletion of the LETM1 gene (12).
We report here on the mitochondrial localization of the human LetM1 protein and of its yeast homologs, Yol027p and Ypr125p (Mrs7p), on their functional homology, and on the effects resulting from the disruption of the yeast genes on mitochondrial functions. The results indicate a role of Yol027p in mitochondrial K ϩ homeostasis. As compared with wild-type mitochondria, mutant yol027⌬ mitochondria exhibit severely reduced potassium acetate (KOAc)-induced swelling, which is indicative of a lack or reduction in K ϩ /H ϩ -exchange activity. We discuss the possibility that YOL027 encodes either the K ϩ /H ϩ exchanger itself or an essential cofactor thereof.
Hemagglutin (HA)-tagged and Green Fluorescent Protein (GFP)tagged Genes-A transposon-tagged YOL027c-containing DNA fragment (15) was used to replace the chromosomal copy of YOL027c in strain GA74 -1A. Upon Cre-mediated recombination, a variant of the YOL027c gene was obtained which had a triple HA-tag inserted inframe after codon 469. This HA-tagged version of the YOL027c gene had no apparent phenotypic effect on the growth of the mutant strain compared with the isogenic wild-type strain.
The YOL027c gene sequence (nucleotides Ϫ426 (relative to the start codon) to ϩ1721) was PCR-amplified from W303 genomic DNA by use of an oligonucleotide 5Ј primer carrying a natural SacI site and a 3Ј primer that introduced a PstI site upstream of the stop codon. The SacI-PstI fragment of this product was inserted into YCp33-HA vector, resulting in plasmid YCp-YOL027-HA.
YPR125 coding sequence (1356 nucleotides from 1 to ϩ1356) was PCR-amplified from the same yeast strain, introducing recognition sites for EcoRI and SalI, followed by insertion of this fragment into the EcoRI and SalI sites of the centromeric vector pUG35. The resulting plasmid pUG35-YPR125-GFP expressed the YPR125 gene under the MET25 promoter and in-frame with the GFP coding sequence following at its 3Ј end. The YEp-YPR125 construct (YEp-MW7) had been cloned by Waldherr et al. (10).
To clone the human LETM1 cDNA, SuperScript II reverse transcriptase (Invitrogen) and an oligo(dT) primer were used for first-strand synthesis on poly(A)-enriched template RNA isolated from a PA-1 human teratocarcinoma cell line. PCR fragments covering the entire coding sequences (from nucleotide 1 to ϩ2590) were amplified, and the purified PCR product was cloned to the pGEM-T vector (Promega), and a C-terminal HA-tag was added by PCR. The PCR product was digested with XhoI and HindIII and cloned into the corresponding sites of the yeast expression vector, pVT103-U. An XhoI-EcoRI PCR fragment was cloned into the pEGFP-N1 mammalian expression vector, thus creating an in-frame fusion with the enhanced GFP (EGFP) sequence carried on the vector.
Gene Deletion-Complete disruptions of YOL027, MRS2, and MRS7 were performed according to the one-step replacement protocol described in Ref. 16 in the diploid yeast strain W303 and the haploid DBY747. Disruption of YOL027 resulted in a deletion of 1702 nucleotides (from the start codon to nucleotide Ϫ19, relative to the stop codon) of the YOL027 open reading frame (named yol027⌬ mutant). Spores derived from this diploid strain were found to be viable. A disruption of the same size then was obtained in DBY747 (haploid). Disruption of the open reading frames MRS2 (mrs2⌬) and MRS7 (mrs7⌬) resulted in deletions of 1218 nucleotides (from nucleotide Ϫ49 relative to the start codon to nucleotide Ϫ243 relative to the stop codon) and of 1356 nucleotides (from the start to the stop codon). 2 W303 yol027⌬mrs2⌬ and yol027⌬mrs7⌬ double-mutant strains were then obtained by crossing the yol027⌬ strain with the mrs2⌬ or ypr125⌬ strain, respectively. Diploids were sporulated, and the haploid double mutants were identified among the meiotic progeny by screening for the appropriate combination of disruption markers.
Isolation and Subfractionation of Mitochondria-GA74 -1A were grown in lactate medium (17), W303 cells in complete YPGal medium or synthetic S-galactopyranoside (S-Gal) medium containing 2% galactose. Strain DBY747 was cultivated in YPD medium. For ion-influx meas-urements, cells were grown to stationary phase; for all other applications, cells were harvested at A 600 of 1. Mitochondria were prepared according to Ref. 18 and suspended in breaking buffer (0.6 M sorbitol HEPES-KOH, pH 7.4) prior to further use. Na 2 CO 3 extraction of proteins from membranes and mitoplast preparation was as described in Refs. 20 and 18, respectively.
Yeast mitochondria obtained by differential centrifugation were diluted to a protein concentration of 10 mg/ml with 10 mM HEPES-Tris-Cl, pH 7.4, and a final osmolarity of 0.1 M. After a 20-min incubation on ice, the resulting mitoplasts were collected by centrifugation (40,000 ϫ g for 10 min at 4°C). To obtain submitochondrial particles (SMP), mitoplasts were resuspended in sucrose buffer (250 mM sucrose, 10 mM Tris-Cl, pH 7.4). The mitoplast suspension was sonified for 3 min with maximum intensity in a Bandelin sonicator UW70/GM70. After removing unbroken mitochondria (10-min centrifugation at 10,000 ϫ g), SMPs were collected by centrifugation at 100,000 ϫ g for 1 h and resuspended in 1 ml of sucrose buffer.
Antibodies used for immunodetection were as described in Ref. Confocal Fluorescence Microscopy-Cells transformed with plasmids expressing GFP fusion proteins were stained with 10 M rhodamine B hexyl ester (Molecular Probes) or 25 nM Mito-Tracker red chloromethyl-X-rosamine and examined by laser confocal microscopy using a Leica TCS4D laser confocal microscope. GFP and rhodamine B were excited by 488 and 543 nm laser lines, respectively, and detected simultaneously at their emission maxima. Mitochondrial polarization was observed by laser confocal microscopy as described in Ref. 3.
Electron Microscopy-W303 cells were grown at 28°C in YPGal to an A 600 of 1.2, fixed for 30 min in 3.7% formaldehyde, spheroblasted with zymolyase at 0.5 mg/g of cells, and washed in phosphate-buffered saline. Spheroblasts were pelleted and resuspended in 2% glutaraldehyde in 0.15 M SorensenЈs buffer (pH 7.4) for postfixation overnight at 4°C. Subsequently, the cell suspensions were filled into cellulose tubes (200 m in diameter), infiltrated with 1% OsO 4 for 1 h, dehydrated in ethanol, and embedded in epoxy resin Agar 100 (Agar Scientific Ltd, UK). Thin sections were cut on a Reichert Ultracut S microtome, mounted on copper grids, and contrasted by uranyl acetate and lead citrate. Grids were examined at 60 kV using a JEM-1210 electron microscope (Jeol Ltd., Japan). Although the size variation among these homologs is high, lower eukaryotes, animals, and plants have at least one predicted transmembrane domain (Fig. 1A). In addition, most proteins in animals and plants have one or two predicted EF-hand calcium-binding domains in their C-terminal extensions, and the mammalian ones contain a leucine zipper region (Fig.  1A), as first noted in Ref. 11.

LetM1p, YOL027p, and Ypr125p Are Members of a Novel Eu
Full-sequence alignments of homologs from plant, human, and yeast ( Fig. 1B) reveal that members of this new protein family are highly conserved in their middle parts (about 40% amino acid identity). The region predicted to contain a transmembrane domain is particularly well conserved (TM, boxed in Fig. 1B). Three prolines within the putative ␣ helical transmembrane sequence (Fig. 1C) are remarkable. Prolines, forming molecular hinges, have been observed repeatedly in transmembrane ␣ helices of proteins, notably in ion channels and G protein-coupled receptors (21).
LetM1p and Ypr125p Localize to Mitochondria-The human LETM1 gene and the yeast YPR125 gene were C-terminally tagged with the GFP-epitope. The LetM1-GFP fusion protein was transiently expressed from the vector pEGFP-N1 in the mouse NIH/3T3 embryonic fibroblast cell line. Fluorescence confocal microscopy revealed the co-localization of the GFP fluorescence with Mito-Tracker fluorescence of mitochondria ( Fig. 2, a-c). When expressed under control of the methionine promoter from a yeast low-copy plasmid (pUG35), the Ypr125-GFP fusion protein co-localized with Mito-Tracker fluorescence, visualizing a distinct tubular network typical of yeast mitochondria (Fig. 2, d-f).
Yol027p Is an Integral Protein of Mitochondrial Inner Membrane-A YOL027-HA allele (triple HA tag C-terminally fused to the YOL027 open reading frame and inserted at the chromosomal locus; see "Experimental Procedures") was used to determine the subcellular localization of Yol027p by cell fractionation and immunoblotting. Total cell content (T), post-mitochondrial supernatant (C), and mitochondrial (M) fractions were separated by SDS-PAGE and analyzed by immunoblotting (Fig. 3A, lanes T, C, M). The cytosolic fraction was characterized by the presence of hexokinase Hxk1p, a soluble protein. Yol027-HAp was found exclusively in the total cell content and mitochondrial fractions, as were the ADP/ATP carrier Aac2p, an integral protein of the inner membrane, and the ␤ subunit of the F1 ATPase, F1␤, a protein associated with the matrix side of the inner membrane.
Treatment of mitochondria by alkaline sodium carbonate (20) solubilized the membrane-associated ATPase subunit F1␤ (Fig. 3A, lane SN), but not the integral membrane protein Aac2p (Fig. 3A, lane P). Yol027-HA protein also stayed in the pellet fraction, thus qualifying it as an integral membrane protein. Cell fractionation, sodium carbonate extraction, and immunoblotting also revealed that Ypr125-GFP and LetM1-GFP behaved as integral proteins of a mitochondrial membrane (data not shown).
To further determine to which of the two mitochondrial membranes Yol027-HA localizes, whole mitochondria, mitoplasts, and SMPs of a yol027⌬ mutant strain expressing a C-terminally HA-tagged Yol027 protein from a single-copy plasmid were obtained, and the accessibility of their proteins by proteinase K was studied (Fig. 3B). In whole mitochondria, all tested proteins were protease resistant, except Tom70p, an outer membrane protein protruding to the surface. Upon disintegration of the membranes by Triton X-100, all proteins were digested by proteinase K, showing that none of them was intrinsically protease-resistant.
Mitoplasts were characterized by (i) the absence of Tom70p, pointing to an efficient removal of the outer membrane, (ii) by protection of the matrix-sided integral membrane protein Tim44 from degradation by proteinase K, (iii) by the degradation of Yme1p, an inner membrane protein with domains exposed to the intermembrane space and the matrix, and (iv) shortening of Aac2p, the ADP/ATP carrier, an inner mitochondrial membrane protein partially exposed to the outside of mitoplasts. Mitoplasts contained Yol027p, but in a proteinase K-resistant form, which implies that no part of this protein protrudes to the intermembrane space to such an extent that it is rendered protease-sensitive.
Sonication of mitoplasts is known to result in the formation of SMPs with a majority of inside-out vesicles (22). Consistently, we found that Tim44p became protease-sensitive, whereas Aac2p lost its protease-sensitivity. The presence of Yol027-HA in these SMPs confirmed its nature as a membrane protein, and its protease-sensitivity indicated that it was exposed to the surface of the SMPs. This change in protease sensitivity of Yol027-HA and Aac2p indicates that sonication of mitoplasts under the conditions used here led to a very large fraction of inside-out particles, allowing the conclusion that the C terminus of Yol027p is located in the mitochondrial matrix.
Disruption of the YOL027 Gene-To investigate the function of Yol027p, the YOL027 coding sequence was replaced by the HIS3MX6 cassette in the diploid yeast strain W303 (see "Experimental Procedures"). After sporulation of the resulting heterozygous strain and tetrad dissection, yol027⌬ spores were found to exhibit reduced growth on non-fermentable carbon sources (YPEG) at 28°C and nearly no growth at 37°C (Fig. 4) and at 18°C (data not shown). Fermentative growth of the mutant (on YPD) was also reduced, as compared with that of the isogenic wild-type (Fig. 4). When grown on glucose containing media, yol027⌬ strains were mitotically unstable, throwing off rho Ϫ cells (having macro-deletions in mitochondrial DNA) at a moderate rate (data not shown).
Disruption of YPR125 had no apparent phenotype. Disruption of both YOL027 and YPR125 (yol027⌬ ypr125⌬ mutant) led to a phenotype indistinguishable from the one exhibited by the yol027⌬ mutant (data not shown). Because YOL027 and YPR125 are multicopy suppressors of the mrs2⌬ petite phenotype, defective in mitochondrial Mg 2ϩ influx (3, 10), we also investigated the phenotypes of a yol027⌬ mrs2⌬ double mutant. This mutant was unable to grow on non-fermentable substrate at any temperature and proved to be rho o (devoid of mitochondrial DNA; data not shown). Simultaneous deletion of YOL027 and MRS2 thus results in a more pronounced (synthetic) growth defect than single deletions of each of these two genes.
Functional Homology of Yeast and Human LetM1p-To find out if the human LetM1p is a functional homolog of Yol027p, we transformed a yol027⌬ strain with a plasmid expressing the LETM1 gene from the strong, constitutive ADH1 promoter on a multicopy plasmid ((LETM1)n). As a control, the strain was also transformed with the empty plasmid and a plasmid containing the YOL027 coding region. As shown in Fig. 4, expression of (LETM1)n restored growth of the yol027⌬ mutant, although not as well as expression of (YOL027)n. Apparently, LetM1p is targeted to the yeast mitochondria and can functionally replace its Yol027 homolog. The yeast homolog YPR125, expressed from a multicopy plasmid, also restored growth of the yol027⌬ mutant strain (Fig. 4).
Mg 2ϩ and Ca 2ϩ Influx into Wild-type and Mutant yol027⌬ Mitochondria-Partial suppression of the mrs2⌬ phenotype by (YOL027)n or by (YPR125)n suggested to us that these two proteins might be involved in mitochondrial cation homeostasis. Comparing influx of Mg 2ϩ and Ca 2ϩ into isolated mitochondria (3), we observed a considerably reduced influx of both Mg 2ϩ and Ca 2ϩ into mutant yol027⌬ mitochondria as compared with wild-type mitochondria (Fig. 5, A and B). Although an increase in external Mg 2ϩ or Ca 2ϩ elicited an initial rapid response, influx quickly ceased, leading to steady-state plateau Identical and conserved amino acids are highlighted in black and gray, respectively. The single predicted transmembrane domains of the proteins are boxed (continuous lines). The C-terminal part of the sequences contains putative EF-hand Ca 2ϩ -binding sites (box, dotted lines) and a putative leucine zipper motif (asterisks). Both have originally been noted for LetM1p (11), and they are not conserved throughout all of the organisms. C, multiple alignment of the exceptionally well conserved transmembrane domain of YOL027 with representatives of its orthologs from other eukaryotic organisms (A). The ␣-helical region is indicated by a bar. The conserved proline residues and a glutamic acid residue within this domain are highlighted. levels considerably lower in yol027⌬ than in wild-type mitochondria (Fig. 5, A and B). This result indicates to us that the Mg 2ϩ and Ca 2ϩ transport systems are active, but saturation of influx is reached at comparatively low intramitochondrial cation concentrations.
As shown by Ref. 3, the driving force for Mg 2ϩ uptake by Mrs2p is the internally negative membrane potential ⌬⌿ of about Ϫ150 mV in mitochondria. We speculated that the absence of Yol027p might result in a reduced ⌬⌿ and, hence, reduced Mg 2ϩ and Ca 2ϩ influx, whereas overexpression of Yol027p might have increased ⌬⌿ and thus improve Mg 2ϩ influx in mrs2⌬ (by so far unknown pathways). In fact, addition of the exogenous cation/H ϩ exchanger nigericin, which is known to enhance ⌬⌿ in respiring mitochondria, was found to stimulate Mg 2ϩ influx into yol027⌬ cells to a considerable extent (Fig. 5A).
Effects of yol027⌬ Mutation on Mitochondrial ⌬⌿ and K ϩ Concentrations-To determine the ⌬⌿ of mitochondria isolated from wild-type and mutant yol027⌬ cells, we used JC-1, a fluorescent imidazole cyanine dye that stays monomeric at low

FIG. 2. Subcellular localization of LetM1 and Mrs7p by fluorescence microscopy.
In vivo co-localization of GFPtagged LETM1 and Mito-Tracker red chloromethyl-X-rosamine in mitochondria of mouse NIH/3T3 cells (a-c) and of GFP-tagged YPR125 and rhodamine B hexyl ester in mitochondria of yeast W303 cells (d-f). The NIH/3T3 mouse fibroblast (a-c) cells were transiently transfected with the pEGFP vector carrying the human LETM1 gene tagged with EGFP at its C terminus. W303 cells expressing Cterminally GFP-tagged YPR125 gene from the centromeric plasmid pUG35-YPR125-GFP (d-f) were grown in S-Gal medium at 28°C to log phase and examined by confocal microscopy.

FIG. 3. Subcellular and submitochondrial localization of Yol027p.
A, transformants of the yeast strain GA74 -1A expressing an internally tagged YOL027-HA gene (see "Experimental Procedures") were grown in synthetic media containing 2% lactate. Protoplasts were homogenized (T) and separated into a mitochondrial (M) and a postmitochondrial fraction (C). Purified mitochondria were treated with 0.1 M Na 2 CO 3 and fractionated by centrifugation at 100,000 ϫ g into pellet (P) and supernatant (SN). Proteins were separated by SDS-PAGE and analyzed by immunoblotting using antibodies to the HA epitope, the mitochondrial proteins Aac2 and F 1 ␤, and the cytosolic protein Hxk1. B, W303 yol027⌬ mutant cells expressing a C-terminally HA-tagged Yol07p (from plasmid YCplac33) were grown in S-Gal-urea medium. Intact mitochondria, (M, left panel), mitoplasts (MP, middle panel), and submitochondrial particles (SMP, right panel) were incubated without and with proteinase K (in the indicated concentrations) or proteinase K plus Triton X-100. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to HA, to the inner membrane proteins Tim44, Aac2, and Yme1, and to the outer membrane protein Tom70.
As shown in Fig. 6a, mitochondrial preparations of wild-type yeast cells exhibit red fluorescence, which is consistent with a high ⌬⌿. Yellow and green spots point to heterogeneity of the membrane potential among mitochondrial particles and even within particles, a phenomenon that has previously been described for mammalian mitochondria (23). In contrast to the wild-type mitochondria, mutant yol027⌬ mitochondria exhibited a green fluorescence (Fig. 6b), indicative of low ⌬⌿. Valinomycin (a K ϩ ionophore) and high external KCl concentrations dissipated the ⌬⌿ to the same extent in mutant and wild-type mitochondria, resulting in green to yellow fluorescence (data not shown). Addition of nigericin, an electroneutral K ϩ /H ϩ exchanger, to respiring mitochondria resulted in an equal fluorescence in wild-type and yol027⌬ particles with compact red spots, indicative of the restoration of a high ⌬⌿ (Fig. 6,  c and d). Nigericin thus fully compensated for the absence of the Yol027 protein, which points to a possible defect in K ϩ homeostasis in the yol027⌬ mutant.
By the use of atomic absorption spectrometry of mitochondrial matrix extracts (24), we observed a drastic increase of the potassium content in yol027⌬ mitochondria (230 nmol/mg of protein) as compared with wild-type mitochondria (125 nmol/mg of protein). Mitochondrial contents of other metals/ elements in mutant mitochondria such as magnesium and sodium were modestly increased by about 40 and 10%, respectively (data not shown).
Increased Volume of yol027⌬ Mitochondria-Increased osmolality, resulting from the net uptake of cations, is expected to be compensated for by an influx of water and swelling of the organelle. In fact, yol027⌬ mutant mitochondria proved to be heavily swollen as compared with wild-type mitochondria, both in situ and in vitro. Transmission electron micrographs (Fig.  7A) revealed enlarged yol027⌬ organelles, lacking tubular shaped cristae and other electron-dense material. Laser confocal microscopy (Fig. 7B) of isolated mitochondria also showed that yol027⌬ organelles in vitro are much larger than their FIG. 4. Growth defects of the W303 yol027⌬ mutant and complementation by YOL027 and its homologs YPR125 and LETM1. A yol027⌬ disruptant of strain W303 was transformed either with the plasmid pVT-U103 without inserts (marked yol027⌬), with inserts YOL027 or LETM1, or with plasmid YEp351 with insert YPR125 (marked (YOL027)n, (LETM1)n, and (YPR125)n, respectively). Serial dilutions of transformants and the wild-type strain W303 (marked YOL027) were spotted onto YPD and YPEG plates and grown at 28°C or for 3 and 5 days, respectively, or at 37°C for 3 and 6 days, respectively. wild-type counterparts prepared and kept in the same buffers. Combined with the data presented above, these findings suggested to us that yol027⌬ mutant mitochondria might be defective in K ϩ homeostasis and possibly in K ϩ /H ϩ exchange, which is of prime importance for the control of matrix K ϩ concentration and volume of the organelle.
KOAc-induced Mitochondrial Swelling-Swelling of isolated mitochondria, determined by light scattering, is a widely accepted optical technique to determine monovalent cation transport across the inner mitochondrial membrane (Refs. 25-27, and reviewed in Refs. 1 and 28). When incubated in KOAc, isolated mitochondria rapidly take up the protonated form of acetic acid. Its ionization in the mitochondrial matrix leads to acidification of the matrix and activation of the K ϩ /H ϩ exchange system. In non-respiring mitochondria, this results in the net accumulation of potassium acetate, uptake of water, and swelling of the organelle, which can be measured as a decrease in light scattering or absorbance (2,25).
Mitochondria of the wild-type strain DBY747 used here showed little change in absorption at 540 nm upon addition of KOAc (Fig. 8A). Rapid swelling was observed, however, upon addition of the ionophore A23187 and EDTA, depleting the system of divalent cations. This effect was abolished in the presence of the protonophore CCCP, consistent with the notion that KOAc-mediated swelling is dependent upon a pH gradient activating the K ϩ /H ϩ exchange. The addition of Mg 2ϩ at molar concentrations exceeding those of EDTA resulted in partial inhibition (data not shown). Quinine, a known inhibitor of the K ϩ /H ϩ exchange reaction, as well as DCCD strongly inhibited swelling (Fig. 8A).
This data parallels previous findings on KOAc-induced swelling of mammalian and yeast mitochondria, except that the need for Mg 2ϩ depletion from the matrix of yeast mitochondria was not observed previously (25)(26)(27). The use of mitochondria from dif-ferent yeast strains may explain this minor discrepancy of results. In fact, when using mitochondria of wild-type strain W303, we also observed spontaneous swelling in KOAc, which was poorly enhanced by Mg 2ϩ depletion (data not shown).
Preparations of mutant yol027⌬ mitochondria from either strain (DBY747 or W303), at concentrations similar to those of wild-type cells, exhibited reduced absorbance at resting conditions ( Fig. 8B and data not shown). This indicates that the organelles were swollen prior to the addition of KOAc, which is consistent with the microscopic data presented above. Furthermore the yol027⌬ mitochondria failed to exhibit rapid swelling in KOAc (plus A23187 and EDTA) (Fig. 8B). These results are fully consistent with the notion of a severe reduction in K ϩ /H ϩ exchange activity in yol027⌬ mutant mitochondria.
Expression of LetM1, the human homolog of Yol027, in the DBY747 yol027⌬ mutant strain resulted in a partial restoration of KOAc-dependent swelling of mitochondria (Fig. 8C). Rapid swelling of those mitochondria was similar to that of wild-type mitochondria in that it was sensitive to quinine and to DCCD. This partial restoration of swelling parallels our finding that LETM1 is able to complement the respiratory growth defect of the yol027⌬ mutant (compare Fig. 4) and indicates that the human LetM1 protein is the functional homolog of the yeast Yol027 protein. Their effects upon KOAcinduced swelling of mitochondria suggests a role for both proteins in K ϩ /H ϩ exchange. DISCUSSION Homologs of the human WHSCR2 (Wolf-Hirschhorn Syndrome Critical Region 2) candidate disease gene LETM1 are ubiquitous in eukaryotes (11,12). The genome of the yeast S. cerevisiae harbors two homologs, MRS7 (YPR125) and YOL027. MRS7 was isolated as a multi-copy suppressor of a mutant defective in mitochondrial Mg 2ϩ influx (3,10). Deletion of YOL027 has been shown to cause changes in mitochondrial morphology and suggested a mitochondrial location of the YOL027 gene product (29). A recent report involving GFPtagged versions of LetM1p (30), as well as this study on human LetM1p, yeast Yol027p, and yeast Ypr125p, confirm a mitochondrial location of LetM1p and its yeast homologs.
Disruption of YOL027 (yol027⌬ mutation) is shown here to result in a defect in respiratory growth, which is consistent with its mitochondrial location. This phenotype can be suppressed by over-expression of the human LETM1 gene in yeast, which is indicative of homologous functions of the LETM1 and YOL027 gene products. YPR125 can also suppress the yol027⌬ mutant phenotype, but a ypr125⌬ mutant did not reveal any obvious growth phenotype.
LetM1, Yol027p and Ypr125p are shown here to be integral membrane proteins. This is consistent with the presence of a predicted, highly conserved transmembrane domain in a central part of all members of this family. By fractionation of mitochondria and immunoblotting, we show that Yol027p is a protein of the inner mitochondrial membrane, exposing the C-terminal part of its sequence toward the mitochondrial matrix. Our finding that no part of Yol027p (or Ypr125p) protrudes to the surface of mitoplasts to an extent that it would be protease-sensitive is at variance with the computer prediction of a single transmembrane domain. Only further studies will reveal if Yol027p has a second, so far unrecognized TM domain, or if its amino-terminal sequence is present on the outside of the inner membrane but embedded into this membrane or otherwise protected from protease degradation.
Detailed phenotypic analysis of the yol027⌬ mutant in comparison to its isogenic wild-type disclosed pleiotropic mitochondrial defects, namely an elevated intramitochondrial potassium level, a swollen appearance of the organelle, and a drastically reduced ⌬⌿. All of these phenotypic features of yol027⌬ mutant mitochondria are consistent with an essential role of the YOL027 gene product in mitochondrial K ϩ homeostasis, possibly in the K ϩ /H ϩ exchange system. Full restoration of ⌬⌿ by the addition of the exogenous K ϩ /H ϩ ionophore nigericin to isolated organelles supports the assumption that a lack in K ϩ /H ϩ exchange activity accounts for the mutant phenotypes of the yol027⌬ mutant.
The activity of the K ϩ /H ϩ exchange system can be unmasked by incubation of isolated mitochondria in KOAc. In non-respiring mitochondria, this treatment results in the uptake of acetic acid, acidification of the matrix, and activation of K ϩ /H ϩ anti-port, with a net increase of [K ϩ ] i and swelling of the organelle (25). Although mammalian mitochondria exhibit rapid swelling only upon prior depletion of divalent cations, yeast mitochondria have been reported to swell spontaneously (26,27).
Mitochondrial preparations of the wild-type yeast strain W303 exhibited spontaneous rapid swelling in KOAc (observed as a decrease in absorbance at 540 nm; data not shown), whereas mitochondria of the wild-type strain DBY747 responded to KOAc addition with a minor change in absorbance (compare Fig. 8) and showed rapid swelling only upon Mg 2ϩ depletion. Thus, concerning their Mg 2ϩ sensitivity of swelling, mitochondria of the latter yeast strain behave similarly to those of mammalian cells. As observed here, rapid KOAc-induced swelling was abolished by quinine or DCCD. This parallels previous studies and is likely to result from K ϩ /H ϩ exchange activity.
Mutant yol027⌬ mitochondria failed to show rapid KOAcinduced swelling. However, swelling was restored to a considerable extent by expression of human LETM1 in yol027⌬ mutant cells. These data support the hypothesis that a lack of Yol027 proteins results in a defect in mitochondrial K ϩ /H ϩ exchange activity. A lack of this activity is sufficient to explain the phenotypic effects observed with yol027⌬ mitochondria: they have highly increased matrix K ϩ content (nearly 2-fold in yol027⌬ mitochondria as compared with wild-type mitochondria), which we interpret as resulting from uncompensated K ϩ leakage in yol027⌬. Increased osmolarity is accompanied by an increase in mitochondrial volume (reviewed in Ref. 28). By using electron microscopy and confocal fluorescence microscopy of whole cells and isolated mitochondria, we observed drastically altered mitochondrial volume and shape in the yol027⌬ mutant, which is consistent with the predicted increased mitochondrial volume as a consequence of elevated K ϩ content. We also found that mutant mitochondria have lower membrane potential than those of isogenic wild-type cells. This may result in part from the observed increase in matrix K ϩ content in yol027⌬ cells. Additionally, the disturbed cation homeostasis may have affected the activity of the respiratory chain or other proton extrusion systems of the organelle. Finally, the exogenous cation/H ϩ ionophore nigericin was found to restore ⌬⌿ in yol027⌬ mutant mitochondria in vitro, presumably by replacing the endogenous K ϩ /H ϩ exchanger.
The data presented here are consistent with an essential role of the Yol027/LetM1 proteins in mitochondrial K ϩ /H ϩ exchange. We cannot discriminate whether Yol027/LetM1 constitutes the exchanger itself or a regulatory factor associated with it. The exceptionally high sequence conservation in the single transmembrane domain might be in favor of a role in transport. Yet, an exchanger with just one transmembrane domain would be unprecedented.
This defect in K ϩ homeostasis of yol027⌬ mutant yeast cells causes a pronounced defect in growth on non-fermentable substrate. A weaker effect upon fermentable substrate, indicating that the absence of the YOL027-encoded protein did not only affect mitochondrial energy conservation systems, but also mitochondrial functions relevant for other processes, such as protein import into the organelles (31), which is dependent upon ⌬⌿. In addition, this single deletion causes instability of mitochondrial DNA. These findings clearly reveal an important role of Yol027/LetM1 in mitochondrial physiology, consistent with their proposed prominent role in K ϩ extrusion from the organelles. Our finding of mitochondrially defective, but viable, yol027⌬ (as well as yol027⌬mrs7⌬) mutant yeast cells indicates that a total breakdown of the mitochondrial K ϩ homeostasis is being prevented in the absence of Yol027 (and its homolog Mrs7/Ypr125) by the activity of other factors.
Knock-down of LetM1p activity seems to severely hamper the development of Caenorhabditis elegans embryos and larvae. 3 Reduction in LetM1 thus seems to have a more serious effect upon the life or development of a multicellular organism than upon that of a unicellular yeast. Provided that the correlation of the hemizygous deletion of LETM1 correlates with the classical Wolf-Hirschhorn syndrome phenotype in humans (12), a change in the LETM1 gene dose seems sufficient to provoke a neuromuscular defect. This result follows the pattern of many reports showing that (i) haploinsufficiency can have dramatic effects on human health, and (ii) neuromuscular disease phenotypes result from mitochondrial dysfunction (32).