Mitochondrial genome integrity mutations uncouple the yeast Saccharomyces cerevisiae ATP synthase

Abstract

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the alpha-, beta-, and gamma-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the gamma-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk.

Figure 1. Color sectoring of the yeast strains containing the mgi mutations

The images show yeast colonies of the wild type (WT) and mutant strains (as indicated) after growth on YPD medium. There are two image plates associated with each strain: one showing the entire culture plate and the second showing the colonies. The ade2 mutation in the strains is responsible for the red color. The absence of the red (white) is due to the petite mutation (see text). The pink color is representative of a defect in oxidative phosphorylation without loss of mitochondrial DNA.

Figure 2. Growth phenotype of the yeast strains with the mgi mutations

The wild type and mutant yeast strains were grown on YPD and YPG at 30 °C at 3 dilutions, as indicated. The haploid cells were mated to the wild type strain, K289-3A (21), and a diploid cell selected and tested for growth in the same manner. Thus, the diploid strain is heterozygous, whereas the haploid strain is homozygous, for the mgi mutation.

Figure 3. In vivo staining of the mitochondrion as a measure of the membrane potential

There are three image plates for the wild type, a mutant strain containing the αN67I mutation, and a cytoplasmic petite mutant strain. The first plate is an epifluorescence image of the cells after staining with Rhodamine 123. The second plate is a phase-contrast image of the same view. The final plate represents the data obtained after fluorescent-activated cell counting. Notice the log scale of the abscissa. In the final plate, the area under the curve with the fluorescence height is listed in Table 2, fourth column.

Figure 4. Representation of the position of the position of the mgi residues in the structure of the yeast F₁-ATPase

A, this is a stripped down view of the yeast F₁-ATPase with the α-(salmon), β-(slate), and γ- (yellow), δ-(gold), and ∊-(purple) subunits. Only pertinentregions of the α-and β-subunits are shown. The mgi residues are colored in red, blue, and green and shown as spheres.B–D, enlargement areas of interest to illustrate the interactions of the residues in the mgi complementation group. The residues and subunits are labeled as indicated. This figure was made using PYMOL (47).