A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis

Abstract

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.

Figure 1.

A protein sequence alignment of putative transmembrane regions in Mdm31-related proteins. Conserved serines, threonines, and acidic residues are indicated by a solid background. Numbers following the species names represent GenBank sequence identifiers.

Figure 2.

Characterization of W303-1B (wt), Δmdm31 (Δ31), Δmdm32 (Δ32), and Δmdm31Δmdm32 (ΔΔ) strains. (A, top) Whole-cell oxygen consumption of cells grown in YPE measured by a Clark-type electrode. Arrows indicate the addition of CCCP to the electrode chamber. (Bottom) Rates of respiration calculated before (open bars) and after (shaded bars) addition of 10 μm CCCP. (B) Frequency of spontaneous, ethidium-bromide- and nigericin-induced petite colony formation in the strains. Data are represented as the mean ± average deviations (error bars). (C) Growth of the strains on YPE plates in the absence or presence of the compounds is indicated at the bottom. Numbers to the left represent the approximate number of cells in each drop. The plates were photographed after 5 days of incubation at 30°.

Figure 3.

Intramitochondrial potassium and magnesium concentrations (nanomoles per milligram of protein) obtained by atomic absorption spectroscopy measurements in mitochondria isolated from the following strains: W303-1B (wt, [K] = 117 ± 26, [Mg] = 42 ± 10); Δmdm31 (Δ31, [K] = 130 ± 17, [Mg] = 72 ± 30); Δmdm32 (Δ32, [K] = 145 ± 35, [Mg] = 82 ± 25); and Δmdm31Δmdm32 (ΔΔ, [K] = 134 ± 18, [Mg] = 134 ± 29). Data are represented as the mean concentration ± average deviations.

Figure 4.

Analysis of mitochondrial morphology by electron microscopy. Electron micrographs of cross sections of permanganate-fixed (A) W303-1B and (B) Δmdm31 cells. Mitochondria are indicated by open arrows. Nuclei (n) and vacuole (v) are also indicated. Bar, 1 μm. The mitochondria in Δmdm31, Δmdm32, and Δmdm31Δmdm32 cells exhibited similar morphology (not shown). (C) The quantitative analysis of mitochondrial cross-section areas in the strains W303-1B (wt, n = 60), Δmdm31 (Δ31, n = 20), Δmdm32 (Δ32, n = 70), and Δmdm31Δmdm32 (ΔΔ, n = 23). Data are represented as the mean area ± average deviations (error bars).

Figure 5.

Passive swelling of mitochondria in isotonic potassium acetate. Light absorbance time scans of mitochondrial preparations from W303-1B (wt), Δmdm31 (Δ31), Δmdm32 (Δ32), and Δmdm31Δmdm32 (ΔΔ) strains. At ∼30-sec intervals (indicated by arrows), valinomycin and CCCP were added as indicated.

Figure 6.

Morphological differences between wild-type and mutant mitochondria. Mitochondrial morphology was visualized by light microscopy following DASPMI staining in the following strains grown in YPD in the absence (top row) or presence of 30 mg/liter nigericin (bottom row): W303-1B (wt), Δmdm31 (Δ31), Δmdm32 (Δ32), and Δmdm31Δmdm32 (ΔΔ).