## 2 Results
### Respiratory Activity of E. magnusii Yeast Mitochondria  
We designed a unique method for mitochondria isolation modified especially for the yeast. The test for fraction contamination included electron microscopy of the organelles and the polarographic assay of the mitochondria function: (1) the electron microscopy images of the _E. magnusii_ yeast (Figure S1 in the Supplementary Section) showed that the mitochondria fraction was pure and free from any inclusions and other organelles. Moreover, the mitochondria in the photos had a regular structure with well-developed cristae and intact outer and inner membranes; (2) the mitochondria could oxidize the respiratory substrates (pyruvate+malate) (Figure S2A in the Supplementary Section) and generate a high membrane potential without any co-factors application (NAD(P)H, cytochrome c) (Figure S2B in the Supplementary Section); (3) the assay of the mitochondria activity showed no endogenous respiration (without any substrate application), indicating neither other membranes nor enzymes (Figure S2A (a) in the Supplementary Section); (4) the mitochondria demonstrated high respiratory rates and phosphorylation, ADP/O ratios close to the theoretically expected maxima (Table S1 in the Supplementary Section) upon oxidation of NAD-dependent substrates. It says about good mitochondria integrity after the isolation. We could detect the activity of adenilate kinase, which is in the inter-membrane mitochondria space and synthesizes ADP from AMP and ATP (Figure S2C in the Supplementary Section). The enzyme cannot work if the membrane is damaged. However, its activity was observed even in the hypotromic incubation medium. We are also sure that the mitochondria fraction was pure because we modified the method of isolation of the mitochondria, especially, to bind and remove the lipid fraction from the suspension. In the mitochondrial isolation and washing medium, we use 1% bovine serum albumin (FAs free fraction V), which binds FAs in the medium. Moreover, the high degree of mitochondria coupling indicates no lipid contamination, as any lipid compounds lead to powerful uncoupling of respiration and phosphorylation [24]. We get live mitochondria with respiratory controls close to the maximum that is impossible if the fraction is contaminated (Table 1, Table S2 in the Supplementary Section). Changes in respiratory activity is the main indicator of the cellular energy status. The mitochondria of most plants, fungi, and yeasts possess an alternative pathway of electron transport that is induced if the main (cytochrome) pathway is inhibited by KCN, azide, or antimycin A [25; 26]. The electron flux is switched off at the reduced ubiquinone site and is specifically inhibited by hydroxamic acid derivatives. The dynamics of the oxygen uptake rate by _E. magnusii_ mitochondria showed that it varied significantly during the growth and ageing of the culture (Table 1). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{4}{c}{**Respiration Rate, ng-Atom Consumed O per 1 mg of Protein**} \\ \cline{2-6}
**Time, h** & & & & **+KCN *** & **+KCN + SHAM*** \\
**V4\({}_{1}\)*** & **V3 *** & **V4\({}_{2}\)*** & **Inhibition** & **Inhibition** \\  & & & & **(\% of Control)** & **(\% of Control)** \\ \hline
17 & 19.6 \(\pm\) 0.63  & 16.95 \(\pm\) 1.23  & 100 & 100 \\
44 & 27.9 \(\pm\) 1.68  & 87.26 & 100 \\
48 & 70.19 \(\pm\) 1.68  & 88.79 & 100 \\
96 & 140.0 \(\pm\) 14.14  & 72.68 & 100 \\
168 & 60.85 \(\pm\) 4.6  & 72.68 & 100 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Respiratory activity and the inhibitory analysis of the _E. magnusii_ yeasts mitochondria in different growth phases upon ageing. The respiration rate in the state 4 (initial respiration) was minimal at the logarithmic growth stage, slightly increased at the 24-h stage, increased threefold in the 48-h phase, and reached its peak by 96 h of growth. The respiration rate of mitochondria in the third state (phosphorylation after the ADP addition) was also altered, similar to that in the second one.