New complexes containing the internal alternative NADH dehydrogenase (Ndi1) in mitochondria of Saccharomyces cerevisiae

Abstract

Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

Perturbation of the yeast mitochondrial lipidome and associated membrane proteins following heterologous expression of Artemia-ANT

“…No significant differences were found for the in-gel activities of Nde1, Nde2 and Ndi1 (belonging to pseudo-complex I) and of complex II among the ArANT-expressing vs Aac2-expressing yeast mitochondria, normalized to the amount of loaded protein deduced by Coomassie staining shown in upper-right panel A (see numbers indicated within upper-left and upper-mid panel A). Although the bands corresponding to pseudo-complex I seem diffuse, this is not unexpected as several of its subunits bind to other subunits of the respiratory chain forming supercomplexes in yeasts 32 , 33 ; thus, pseudo-complex I activity will be dispersed as a function of binding of its components to other subunits with a variable molecular weight. Lower in-gel complex III and IV activities were observed in the ArANT-expressing strain, compared to the Aac2-expressing strain (normalized to the amount of loaded protein deduced by Coomassie staining, bottom-right panel A).…”

Alternative NAD(P)H dehydrogenase and alternative oxidase: Proposed physiological roles in animals

“…The RS can be branched or modified at points of ubiquinone reduction and ubiquinol oxidation. The branching at the point of ubiquinone reduction is mediated by alternative (or type II) NADH dehydrogenases (NDH2s), single-or oligo-subunit enzymes which catalyse the same reaction as complex I but do not pump protons across the IMM (Melo et al, 2004;Rasmusson et al, 2004;Iwata et al, 2012;Matus-Ortega et al, 2015). These enzymes are not inhibited by rotenone, a classic inhibitor of complex I.…”

Regulation of the Stress-Activated Degradation of Mitochondrial Respiratory Complexes in Yeast

“…The advantage of the formation of mitochondrial supercomplexes could be the favored electron transfer from different respiratory subcomplexes to common acceptors such as ubiquinone in the case of CI and CIII and the control of ROS production ( Lapuente-Brun et al, 2013 ; Maranzana et al, 2013 ; Genova and Lenaz, 2015 ). In fact the CI/CIII containing complex is one of the most prominent supercomplexes found in higher eukaryotes and yeast Ndi1 CI equivalent has been recently found in complexes with CIII ( Matus-Ortega et al, 2015 ). The supercomplex organization of the mitochondrial electron transport chain could additionally enable the cell to selectively control the quality of modules of the respiratory chain during active respiration or after mitochondrial damage.…”