Novel components of an active mitochondrial K(+)/H(+) exchange

Abstract

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.

FIGURE 1.

Multicopy suppressors and their growth effects on mdm38Δ. Effects of YDL183c, PIC2, MRS3, and MRS7 on the nonfermentative growth of mdm38Δ cells are shown. W303 mdm38Δ mutant cells containing an empty vector or a vector overexpressing YDL183c, PIC2, MRS3, or MRS7 and wild-type (WT) cells were spotted onto YPD or YPG plates and grown at 28 °C for 3 or 5 days, respectively.

FIGURE 2.

Mitochondrial morphology in function of overexpression of the proteins Pic2, Mrs3, Mrs7, or Ydl183c in W303 mdm38Δ mutant cells. Mitochondrial morphology of cells cotransformed with a mitochondrial matrix targeted GFP (pYX232-mtGFP) and the vector without (a) or with the following suppressor genes: PIC2 (c), MRS3 (d), YDL183c (f), and MRS7 (e) were compared with wild-type (WT) cells (b). Cells were grown in galactose-containing medium and analyzed by differential interference contrast (Nomarski) and confocal fluorescence microscopy.

FIGURE 3.

KHE activity of mdm38Δ SMPs in the function of the suppressors Pic2, Mrs3, Mrs7, and Ydl183c. Submitochondrial inner membrane particles were prepared from wild-type and mdm38Δ mutant cells with entrapped K⁺-sensitive PBFI or H⁺-sensitive BCECF. Ratios of K⁺-bound or H⁺-bound to -unbound dyes were recorded at 25 °C at resting conditions and upon the addition of 150 mm KCl. A, shown are the effects on K⁺ and H⁺ fluxes in SMPs upon overexpression of the suppressor genes in W303 mdm38Δ. SMPs were prepared from mitochondria of wild type (WT) (black thin dashed line) or mutant mdm38Δ cells carrying the empty plasmid (gray solid) or the suppressor plasmid containing the genes PIC2 (black dotted line), MRS3 (black bold dashed line), MRS7 (gray square dotted line), or YDL183c (black solid line). B, increase of [K⁺]_i and [H⁺]_i observed in SMPs from DBY wild-type (black dashed line), single mutant mdm38 (gray dashed line), or triple mutant mdm38Δ mrs7Δ ydl183cΔ (black thin solid line) in the absence of nigericin or mdm38 (gray bold solid line) and mdm38Δ mrs7Δ ydl183cΔ (black bold solid line) in the presence of nigericin.

FIGURE 4.

Ydl183cp is a component of the mitochondrial inner membrane. A, Ydl183cp is a member of a novel protein family. Homologous proteins were identified by a BLAST search. A sequence alignment (ClustalW) of Ydl183cp and its homologues in A. thaliana (A.t.) and Neurospora crassa (N.c.) is shown here. Identical amino acids are highlighted in black and similar amino acids in gray. The putative potential N-terminal mitochondrial targeting sequence is marked with a dotted bar and the putative transmembrane domain with a solid bar. B, localization of the Ydl183c-GFP fusion protein analyzed under confocal microscopy. W303 cells expressing C-terminally GFP-tagged YDL183c gene were grown to log phase in galactose containing medium at 28 °C. Mitochondria are labeled with MitoTracker red chloromethyl-X-rosamine. C, subcellular and submitochondrial localization of Ydl183cp. Panel a, W303 cells expressing the Ydl183c-HA fusion protein (YCp-YDL183c-HA, 42 kDa) were grown to log phase in galactose-containing medium. Protoplasts were homogenized and separated into total cell (T), mitochondrial (M), and post-mitochondrial (C) fractions. Equal amounts of protein of subcellular fractions were subjected to SDS-PAGE, and immunodetection with antisera against the HA tag, Hxk1p and Por1p, was performed. Panel b, crude mitochondria (2 mg of protein) were treated with 0.1 m Na₂CO₃ and fractionated by centrifugation at 100,000 × g into pellet (P) and supernatant (SN). Both fractions (100 μg of protein/lane) were subjected to SDS-PAGE and immunoblotted with antisera against HA, Por1p, and F₁β. Panel c, mitoplasts prepared by osmotic shock were separated into supernatant containing the inter-membrane space and pellet. Mitoplasts were aliquoted in equal amounts and incubated with or without proteinase K as indicated. Samples were analyzed by SDS-PAGE and immunoblotted with antisera against the HA tag and against mitochondrial proteins of the inner membrane Tim44p and Yme1p.

FIGURE 5.

Deletion growth phenotypes. A, serial dilutions of DBY. mdm38Δ, mdm38Δ mrs7Δ, mdm38Δ ydl183cΔ, and mdm38Δ mrs7Δ ydl183cΔ mutants were spotted onto YDP and YPG and incubated at the indicated temperatures. Growth on 28, 35.5, and 16 °C was observed after 3, 5, and 8 days, respectively. B, DBY wild-type (WT) and mdm38Δ mrs7Δ ydl183cΔ triple mutant cells expressing an empty control vector (pUG35) or YCp33-MDM38-HA, pUG35-MRS7-GFP, pUG35-YDL183c-GFP, or pVT-U-LETM1-HA. Serial dilutions were spotted onto YPD and YPG plates and incubated for 10 days at 16 °C or 3 or 5 days at 28 and 37 °C on YDP or YPG, respectively. C, effect of nigericin on the nonfermentative growth of DBY747 mdm38Δ single, mdm38Δmrs7Δ, mdm38Δydl183cΔ double, and mdm38Δ mrs7Δ ydl183cΔ triple mutant cells. Serial dilutions of the wild-type and mutant cells were spotted onto YPD and YPG plates containing (+) or not (−) 2 μm nigericin and incubated 10 days at 16 °C and 5 days at 28 °C.

FIGURE 6.

Suppression effect of human HCCR-1. A, sequence alignments of Mdm38, Mrs7, Letm1, and HCCR-1. ClustalW alignments of the amino acid sequences over the homologous regions are shown. Identities are highlighted in black and similarities in gray. Amino acid residues identical over all four sequences are in boldface and boxed. Bar is over the transmembrane domain. B, growth effect of HCCR-1 expression in yeast triple mdm38Δ mrs7Δ ydl183Δ mutants (ΔΔΔ). Wild-type (WT) and triple mutant cells expressing pVTU103 with or without HCCR-1 were spotted onto SD−ura, YPD, and YPG plates and grown at the indicated temperatures for 6, 3, and 6 days, respectively. C, subcellular localization of HCCR-1 in yeast. Yeast triple mdm38Δ mrs7Δ ydl183Δ mutants (ΔΔΔ) expressing HCCR-1 were fractionated into total (T), mitochondrial (M), and post-mitochondrial (C) fractions, and Western blotting was performed.

FIGURE 7.

Mitochondrial and vacuolar morphology in absence of Mdm38p, Mrs7p, and Ydl183cp. Cells were grown to logarithmic phase in galactose (A and B)- or galactose- and raffinose (C)-containing medium. Shown are representative fluorescent and electron microscopy images. A, confocal microscopy analysis of W303 wild-type cells (a), isogenic mdm38Δ (b) and isogenic mdm38Δ mrs7Δ ydl183cΔ triple mutant cells (c) expressing the mitochondrial matrix targeted GFP. Vacuoles were stained with FM4-64. B, electron micrographs of mdm38Δ mrs7Δ ydl183cΔ cells. Panels a and b show the organellar ultrastructure of the triple mutant grown as described above. Whole cells are shown in right panels. The cells display mitochondria with aberrant morphologies (details showing mitochondria are in the left panels). Panel c shows the organellar ultrastucture of cells from the same culture to which nigericin (2 nm) has been added for the last growth generation. Right panel, whole cells; left panel, mitochondrion after nigericin treatment. Bar, 200 nm (left panels) and 1 μm (right panels). C, confocal microscopy analysis of wild-type cells (panels a–d) and mdm38Δ mrs7Δ ydl183cΔ cells (panels e–h) expressing the mitochondrial targeted YFP (panels a and e) to the outer membrane (pHS72). Vacuoles are indicated by FM4-64 (panels b and f). Merged fluorescence is shown in panels c and g. The yellow fluorescence detected indicates the colocalization of mitochondria and vacuoles. Differential interference contrast microscopy of wild-type (panel d) and mdm38Δ mrs7Δ ydl183cΔ (panel h) cells is shown.

FIGURE 8.

KHE activity of mdm38Δ mrs7Δ ydl183c Δ SMPs. [K⁺]-driven changes of [K⁺]_i and [H⁺]_i in submitochondrial inner-membrane particles prepared from wild-type and mdm38Δ mrs7Δ ydl183cΔ mutant cells with entrapped K⁺-sensitive PBFI or H⁺-sensitive BCECF were recorded as described in Fig. 3. A, effect of overexpression of Mrs7p (black square dotted line) or Ydl183cp (black thin dashed line) on [K⁺]-driven changes of [K⁺]_i and [H⁺]_i in DBY triple mutant mdm38Δ mrs7Δ ydl183cΔ SMPs (black solid line) in comparison with wild-type SMPs (black dotted line). B, effect of Mdm38p (expressed from YCp33, bold black solid line) or LETM1 (expressed from pVTU-(bold gray solid line) on [K⁺]-driven changes of [K⁺]_i and [H⁺]_i in DBY mdm38Δ mrs7Δ ydl183cΔ SMPs (black thin solid line) in comparison with wild-type SMPs (bold square dotted line).

FIGURE 9.

Mdm38p, Mrs7p, and Ydl183cp are part of a high molecular weight complex. A, DBY chromosomally Mdm38-His-tagged mitochondria were solubilized with 1.2% Triton X-100. Left panel, one part of the preparation was immediately separated on BN-PAGE. The anti-His antibody recognized three protein complexes of ∼500, <232, and < 40 kDa. Middle panel, other part of the same preparation was used for a further step involving nickel-affinity chromatography. Mdm38-His was recovered as part of a complex of <232 kDa. Right panel shows Mdm38-STrEP after STrEP-affinity chromatography elution separated on BN-PAGE. The anti-STrEP antibody recognized the complexes of <232 and >440 kDa. M, marker. B, DBY mitochondria expressing the chromosomally His-tagged Mrs7 were solubilized as in A and analyzed by BN-PAGE (left panel, lane 1), and parallel fractions were used for further isolation of a Mrs7-His complex by affinity chromatography (left panel, lane 2). Solubilized mitochondrial proteins and elution fractions from the affinity purification were separated on the same BN gel, transferred to a common membrane for Western blotting, and probed with an antiserum against His. DBY mitochondria expressing the chromosomal Mrs7-STrEP were solubilized, affinity-purified, and recovered in complexes of <140, >232, and >440 kDa. C, mitochondria expressing pUG-YDL183c-GFP in different backgrounds as follows: wild-type (lane 1) or mdm38Δ (lane 2) were solubilized with 1.2% n-dodecyl β-d-maltoside. Equal amounts of proteins were separated on BN-PAGE and immunoblotted with an antibody against GFP.

FIGURE 10.

Interaction of Mrs7-His with Mdm38-HA and YDL183c-HA. A, affinity chromatography and preparative BN-PAGE of solubilized mitochondria coexpressing chromosomally His-tagged Mrs7 and extra-chromosomal YCp-Mdm38-HA in different backgrounds as follows: wild-type (WT) (lanes 1 and 2) and mdm38Δ (lanes 3–5). 120 μl (lanes 1, 3, and 5) and 60 μl (lanes 2 and 4) of the eluted fractions were applied to the same gel. Lanes 1–4 were probed with an antibody against His. Lane 5 served for the additional immunodetection with an antibody against HA. M, marker. B, second dimension SDS-PAGE of lane 3. Left panel, the antibody against His recognizes a product of ∼55 kDa corresponding to Mrs7-His. The signal is in perfect agreement with the signals of the first dimension (BN-PAGE). Right panel, immunodetection with anti-HA antibody of the same blot after mild stripping. C, affinity chromatography and BN-PAGE of solubilized mitochondria coexpressing chromosomally His-tagged Mrs7 (lanes 1 and 3) or His-TAP-tagged Mrs7 (lanes 2 and 4) and extra-chromosomal Ydl183-HA (lanes 1–4). Lanes 1 and 2 and lanes 3 and 4 were probed with antibodies against HA and His, respectively.

FIGURE 11.

Interaction of Mdm38p with Mrs7-His and Mrs7-His-TAP. A, affinity chromatography and BN-PAGE of solubilized DBY mitochondria expressing either chromosomally His-tagged or His-TAP-tagged Mrs7. Eluted fractions 1–2 containing Mrs7-His were applied on lanes 1 and 2 and 5 and 6 and eluted fractions 1–2 containing Mrs7-His-TAP on lanes 3 and 4 and 7 and 8. BN-PAGE was performed and followed by immunostaining with an antibody against His (lanes 1–4) and Mdm38p (lanes 5–8). M, marker. B, preparative affinity chromatography and BN-PAGE of DBY mitochondria expressing chromosomally Mrs7-His-TAP prior to second dimension SDS-PAGE. The membrane was first incubated with an anti-His primary antibody (lane 1). Thereafter, the blot was mildly stripped and reincubated with an antibody against Mdm38p (lane 2). C, second dimension SDS-PAGE. Left panel, the blot was probed with the anti His antibody. Right panel, same blot probed with the anti Mdm38p antibody after mild stripping of the membrane. D, suppression effect of Mrs7-His and Mrs7-His-TAP in mdm38Δ. DBY wild type (WT) with YEp112 empty and mdm38Δ with YEp112 empty, MRS7-His, or MRS7-His-TAP were grown overnight. Serial dilutions were spotted onto YPD and YPG plates and incubated at the indicated temperatures.

FIGURE 12.

CoIP of isolated mdm38Δ mitochondria coexpressing YEp-MDM38-HA (72 kDa) and pUG-MDM38-GFP (92 kDa) (A), YDL183-GFP (65 kDa) (B), or AIF-GFP (68 kDa) (C). F, flow-through fraction; B, HA-coated protein A-bound fraction.