*3.2. Hints for Mitochondrial Dysfunction*

− − − − − − − Electron microscopy revealed uneven and swelled mitochondria with barely detectable cristae in patient's tubular epithelial cells (Figure 1C). Histochemical staining of cytochrome *c* oxidase (COX or CIV) and NADH dehydrogenase (CI) activities was strongly reduced in renal tissues from the patient in comparison to control kidney samples (Figure 1D), whereas the activity of succinate dehydrogenase (SDH) was much less diminished. No large rearrangement (deletion) and depletion of the patient's mtDNA were detected (not shown). The mitochondrial genome of the patient was entirely sequenced, which revealed a number of nucleotide changes relative to the reference human mitochondrial genome (Table 4), among which m.3243A>G in mt-tRNALeu, which is the most frequent pathogenic mtDNA allele [38] (Figure 2A). Another point mutation, never reported thus far, was detected in the *MT-ATP6*: m.8909T>C. It is absent in 2704 controls in databases and in 100 age-matched controls from the Nanjing geographic region from which the patient originated (Figure 2B). Pyrosequencing analyses revealed that the *MT-ATP6* variant was homoplasmic in blood, urine sediments (epithelial-like cells detached from tubules) and kidney, whereas the m.3243A>G change was heteroplasmic (50–90%). Defects in mitochondrial translation induced by the m.3243A>G mutation likely explain the poor histochemical

− −

staining of CI and CIV, two complexes of mixed genetic origin, and the much better preservation of the entirely nucleus-encoded SDH complex.


**Table 4.** List of the mtDNA nucleotide changes in the patient relative to the reference sequence of the human mitochondrial genome (http://www.mtdb.igp.uu.se/).

<sup>a</sup> Frequency refers to the occurrence of the detected nucleotide changes in 2704 control individuals except D-Loop.

\* Stands for haplogroup B4b1a nucleotide changes.

The m.8909T>C variant converts a phenylalanine residue into serine at position 128 of human subunit *a* (*a*F128S) of ATP synthase. This residue is highly conserved in a large panel of evolutionary distant species (see below). Therefore, if the m.3243A>G mutation in mt-tRNALeu likely impacted the two mtDNA encoded subunits (ATP6 and ATP8) of ATP synthase, as was observed in previous studies of cells containing this mutation [39–41], we considered that the m.8909T>C possibly affected also the ATP synthase. It would have been difficult to test this hypothesis from patient's cells and tissues because of their mitochondrial genetic heterogeneity. We, therefore decided, as described below, to investigate the consequences in isolation of an equivalent of this mutation on the yeast ATP synthase.

**Figure 2.** Mitochondrial DNA sequencing. Entire sequencing of the mtDNA of the patient identified the well-known pathogenic mutation m.3243A>G in *MT-TL1* (**A**) and a novel variant in *MT-ATP6*, m.8909T>C (**B**). The former was heteroplasmic whereas the latter was homoplasmic in analyzed cells and tissues (blood, urine and kidney). All the nucleotide changes relative to the reference human mitochondrial genome sequence are listed in Table 2.

#### *3.3. Consequences of the m.8909T*>*C Mutation on Yeast ATP Synthase*

Yeast subunit *a* (also called subunit *6* or Atp6) is synthesized as a precursor protein the first ten residues of which are removed during ATP synthase assembly [42]. The phenylalanine residue at position 128 of human subunit *a* that is changed into serine by the m.8909T>C mutation corresponds to the phenylalanine residue at position 145 in the mature yeast protein (155 in the unprocessed form) (see below). A yeast model homoplasmic for the m.8909T>C mutation was created by changing the phenylalanine codon TTC 155 into TCA (see Materials and Methods).
