External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency inYarrowia lipolytica

Abstract

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

Shared evolutionary footprints suggest mitochondrial oxidative damage underlies multiple complex I losses in fungi

“…We found that AOX loss is always related to CI loss (preceding it in 4/6 events), whereas NDH2 duplication is associated with CI loss (preceding 3/6 events) in all but S. bacillaris . This suggests that the ancestral species that lost CI already lacked AOX and had increased alternative NDH2 activity (which could have compensated loss of CI function, as previously described [10,11,56]). In S. bacillaris , the increase in mitochondrial NDH2 activity may be mediated through the expanded, mitochondrially retargeted, NADH oxidase family (figure 2 e ).…”

“…royalsocietypublishing.org/journal/rsob Open Biol. 11: 200362 lacked AOX and had increased alternative NDH2 activity (which could have compensated loss of CI function, as previously described [10,11,56]). In S. bacillaris, the increase in mitochondrial NDH2 activity may be mediated through the expanded, mitochondrially retargeted, NADH oxidase family (figure 2e).…”

“…Accordingly, we predict increased fermentative capabilities and duplicated NDH2 in cases of recent CI loss. In addition, this explains why the retargeting of NDH2 into mitochondria rescues a (otherwise lethal) CI defect [56].

Figure 5Proposed model for the causes and consequences of complex I.

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Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli

“…NDH-2 performs the same catalytic function as respiratory complex I, and its expression was observed to restore the mitochondrial activity in yeast and animals with complex I deficiency. , Therefore, NDH-2 has attracted interest as a key enzyme for the treatment of complex I-related neurodegenerative disorders like Parkinson’s disease . In addition, NDH-2 is the only enzyme with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms, and for that, it has been proposed as a possible new drug target for the rational design of antibiotics. − …”