*2.5. Construction of S. cerevisiae Strain RKY108 (aF145S)*

We used the QuikChange XL Site-directed Mutagenesis Kit of Stratagene to introduce an equivalent of the m.8909T>C mutation (*aF145S*) in the yeast *ATP6* gene cloned in pUC19 [30], utilizing the oligonucleotide 5′ -GGTTTATATAAACATGGTTGAGTATTCTTCTCATT ATCAGTACCTGCTGGTACACCATTACC-3′ . The *atp6*-F145S gene was cloned into the plasmid pJM2 [8]. The resulting plasmid (pRK66) was introduced into mitochondria of the strain DFS160 that totally lacks mitochondrial DNA (ρ 0 ) as described [8] (see Table 2 for complete genotypes and sources of yeast strains). The resulting mitochondrial transformants (RKY109) were crossed to the *atp6*::*ARG8m* deletion strain MR10 [30] to yield strain RKY108. This strain has the MR10 nucleus and the *atp6*-F145S gene in a complete (ρ <sup>+</sup>) mitochondrial genome. The presence of the *atp6* mutation in these clones was confirmed by DNA sequencing. No other change was detected in the *ATP6* gene. The corresponding wild type strain MR6 (WT) is a derivative of strain W303-1B in which the nuclear *ARG8* gene has been replaced with *HIS3* (*arg8::HIS3*) and with the entirely sequenced mitochondrial genome of strain BY4741 [30].


#### **Table 2.** Genotypes and sources of yeast strains.

Yeast genes nomencalure is used in accordance to http://www.yeastgenome.org/help/community/nomenclatureconventions.

#### *2.6. Yeast-Based Drug Assay*

0.05 OD650nm of exponentially growing cells were homogeneously spread with sterile glass beads on a square Petri dish (12 cm × 12 cm) containing solid YPAGly medium. Sterile filters were deposited on the plate and spotted with oligomycin (purchased from Sigma, St. Louis, MO, USA) dissolved in DMSO.
