Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae

Abstract

The phenotypic manifestations of mitochondrial DNA (mtDNA) mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R) or P(R) 454) mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R))), the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S)), mto2(P(S)) and MTO2(P(R))). The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R)) strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

Figure 1. Secondary structure of small rRNA decoding sites in yeast and human mitochondria.

A, secondary structure of E. coli small ribosome rRNA decoding site. B, wild type and P^R mutant forms of yeast 15S rRNA decoding sites, and the base-pair affected by P^R mutation are indicated by arrowheads. C, the corresponding regions of human mitochondrial 12S rRNA are shown as the wild type version and versions containing A1555G and C1494T mutations, respectively.

Figure 2. Growth activities of different yeast strains.

A, Series dilutions of each strain were spotted onto a 2% glucose medium (YPD) and the plate was incubated at 30°C for 72 hours. B, Growth curves analysis of yeast strains in the absence of neomycin in 20 hours. C, Growth activities of each strain when grown on medium containing neomycin after 72 hours incubation. D, Growth curves of strains cultured in YPD containing 32 µg/ml neomycin.

Figure 3. Assay of Oxygen consumption rates.

Cells were harvested in the mid log-phase. The oxygen consumption rate of each yeast strain was measured at the density of 4×10⁵ cells/well by FX-96 oxygraph (SeaHorse Biosciences) in the absence or presence of 32 μg/ml neomycin. The results are shown as means±SD of triplicate.

Figure 4. In vivo staining of the mitochondrion to measure the membrane potential by Rhodamine 123 dyes.

2×10⁶ cells in 1 ml supernatant were incubated with Rhodamine 123 (5 µg/ml) for 20 min at 30°C. Cell pellets were resuspended in 20 µl PBS and visualized with Carl Zeiss 710 LSM microscopy. The relative fluorescence signal of each strain is shown in the right panel.

Figure 5. Northern blot analysis of transcription levels of mitochondrial genes.

A, northern blot analysis of mitochondrial 15S rRNA and 21S rRNA. The nuclear encoded 25S rRNA was hybridized as a internal control. B, quantitative analysis of mitochondrial rRNA transcription levels, where each calculation was based on three independent determinations of each RNA in each yeast strain. C, northern blot analysis of CYTB, COX1, ATP6 and ATP9. D, quantitative analysis of mitochondrial mRNA transcription levels.

Figure 6. Steady-state level of key glycolytic genes.

A, northern blot analysis of HXK2, PFK1 and PYK1 transcription levels. The nuclear encoded 25S rRNA was hybridized as an internal control. B, relative expression levels of these genes were calculated by three determinations. C, translational levels of hexokinase in each strain, tubulin was as an internal control. D, calculation of the relative expression level of hexokinase.

Figure 7. Properties of yeast cells on YPD plate in the presence of glycolytic inhibitor 2-Deoxy-D-glucose (2-DG).

Series dilutions of each strain were spotted on medium and the plate was incubated at 30°C for 72 hours. A, on YPD medium. B, on YPD medium supplemented with 2.5 mM 2-DG. C, on YPD medium supplemented with 2.5 mM 2-DG and 300 µg/ml neomyocin.