For functioning without oxygen as terminal electron acceptor, eukaryotes such as S. cerevisiae have to maintain redox balance without aerobic respiration. Aerobic respiration occurs in the mitochondria, and some eukaryotes such as protists and metazoa have functional mitochondria without respiration, in case of which terminal electron acceptors other than O2 are utilized 34. S. cerevisiae is rather unique among eukaryotes and even among other yeast species in that it can grow fast in the presence as well as in the absence of oxygen. The NADH, produced throughout glycolysis, is consumed in the reaction of pyruvate to lactate or ethanol, end products that are subsequently secreted. Despite a plethora of scientific publications addressing mitochondria and their role in yeast physiology, comparatively little is known about how the absence of oxygen and therefore of respiratory chain activity, affects mitochondrial structure and function. This study focusses on the S. cerevisiae mitochondria proteome with an emphasis on membrane protein complex formation and stability.
4.1 Overall quantitative analysis of the mitochondrial proteome
S. cerevisiae was cultured in chemostats to optimally control growth conditions, using either aerobic or anaerobic environment. The lysates were enriched for the mitochondria by differential ultracentrifugation, whereas anaerobic samples were strictly kept oxygen-free during fractionation 26. However, the commonly applied last step of density centrifugation using sucrose gradients to further purify the mitochondria was omitted for several reasons. This was avoided since the density centrifugation step might have introduced oxygen into the anaerobic samples, which may have resulted in superfluous effects on mitochondrial proteome levels or protein complex composition. Most importantly, earlier studies revealed that the morphology of mitochondria changes upon anaerobic growth conditions, resulting in dissimilarly shaped mitochondria 15, which is not compatible with proper purification of mitochondria using density gradient purification.
We compared the current results using mitochondrial fractionation with our earlier results on the relative quantification of total cellular yeast protein lysates 6. This earlier robust data set contained only 30% mitochondrial proteins, whereas for the current set this improved to 58%, and in addition, the number of reliably quantified mitochondrial proteins increased from 143 to 362. It is thus apparent that our relatively simple protocol used for mitochondria enrichment was proven successful.
Further, comparing the quantified mitochondrial proteins of the current data set with this former data set 6 by Spearman rank (SR) correlation analysis resulted in an SR coefficient of 0.692, which is rather good, considering that the character of both samples, the total lysate and the mitochondrial-enriched fraction, is fairly different. Accordingly, we detected an SR coefficient of 0.486 for the non-mitochondrial proteins.
4.2 Mitochondrial membrane protein complex formation and/or stability under anaerobiosis
We successfully separated and relatively quantified mitochondrial protein complexes using a novel combination of 1-D BN-PAGE, a non-denaturating electrophoresis method that keeps protein complexes intact, and 14N/15N-labeling for anaerobic and aerobic mitochondrial samples for accurate quantification of the relative levels of all protein complex members individually (Fig. 3, Supporting Information Table 3). Usually BN-PAGE is followed by a second dimension, i.e. SDS-PAGE, to separate the complex members based on the overall size, though by using our innovative approach, we obtain a more accurate evaluation of relative protein levels and specific protein interactions, thus a second dimension is no longer needed. These quantification data are very reliable and robust, and it has been shown earlier that S. cerevisiae metabolic labeling with 14N/15N in combination with chemostat culturing provides highly reproducible quantitative data 6, 24. Kolkman et al., have also shown that these protein quantitative data can be validated with Western blot and quantitative PCR 24.
The complex with the highest Mr is the prohibitin complex that is assembled into a membrane-associated ring-shaped supercomplex of approximately 1 MDa, consisting of Phb1p and Phb2p, and acts as a chaperone for newly synthesized membrane proteins 35–37. This complex is also known to be associated with certain m-AAA proteases 37, which were detected with a similar ratio (Supporting Information Fig. 3).
The mitochondrial protein respiratory chain complexes were still present when yeast cells were grown under anaerobic conditions, but their levels were reduced to an average of 55–60% for complex V dimer and monomer, and even to an average of 20–30% for the two complex III/IV supercomplexes, (III)2(IV)2 and (III)2(IV). Moreover, the composition of these complexes is unchanged, since migration patterns of all supercomplex members are highly correlated. S. cerevisiae can grow rapidly under anaerobiosis, whereas it can also adapt quickly when growth conditions are changed back into aerobiosis, which may be facilitated when the respiratory chain complexes are still present in the anaerobic mitochondria 38, 39. Furthermore, although oxygen is not available as final electron acceptor under anaerobiosis, it has been reported that some remaining activity exists in the mitochondrial respiratory chain, suggesting that other electron acceptors are being utilized 38.
A remarkable change for anaerobic mitochondria, however, was found for complex II that showed a significant change in migration for all five complex II proteins Sdh1-4p and Sdh1Bp. The ratio anaerobic/aerobic in the BN-PAGE experiment could only be determined for Sdh1p, which was 1.3±0.1 (Supporting Information Table 3), thus no change in mitochondrial level was detected for this complex II member. Still, the difference in migration points at a difference in complex II composition between aerobic and anaerobic mitochondria, which may be due to the association with other proteins or protein complexes. It has been found earlier through other native electrophoresis experiments that yeast mitochondrial dehydrogenases can form supramolecular complexes 36. A model was proposed that linked the mitochondrial membrane-bound dehydrogenases Gut2p, Nde1p, Nde2p and Ndi1p to a.o. Sdh1p and Fum1p as TCA cycle enzymes that are located close to the membrane 36. Except for Nde1p and Nde2p, we detected all these proteins in our BN-PAGE quantification experiment, all with reduced levels under anaerobiosis, i.e. ranging from 20 to 50% reduction (Supporting Information Table 3), which might explain this change in migration distance within the 1-D BN-PAGE gel. Gut2p is localized in the inner mitochondrial membrane, where it oxidizes glycerol-3-phosphate to dihydroxyacetone phosphate. Together with Gut1p, it acts as a glycerol-3-phosphate shuttle that is responsible for oxidation of NADH under aerobic conditions 40. It has been proposed that under anaerobiosis the aldehyde dehydrogenase Adh3p, collaborates together with the fumarate reductase and the glycerol-3-phosphate dehydrogenase to maintain redox balance 41. We detected down-regulation of Gut2p, which points to reduced participation of the glycerol shuttle in the control of the NAD/NADH redox balance during anaerobiosis. Adh3p, however, was found at higher levels (Supporting Information Table 1), indicating its importance for maintaining the redox balance during anaerobiosis.
Correlation of protein profiles has been successfully used earlier to identify unknown members of organelles 21, 42, 43 and here a variant of this principle was demonstrated by analyzing the protein migration patterns in BN-PAGE. Pearson correlation analysis to the BN-PAGE protein migration profiles of all proteins in the mitochondrial samples revealed clusters of proteins with known interactions (Figs. 4 and 5). Interestingly, by using this approach we could assign protein Aim38p, with unknown function, as possible member of complex IV (Fig. 6). Although further evidence should be obtained that Aimp38p is indeed a complex IV member, this protein was identified as mitochondrial protein 14, whereas deletion of the AIM38 gene leads to impaired growth on non-fermentable carbon sources 44, which points at its involvement in respiration.