In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway

Abstract

In Yarrowia lipolytica, mitochondria contain a branched respiratory chain constituted by the classic complexes I, II, III and IV, plus an alternative external NADH dehydrogenase (NDH2e) and an alternative oxidase (AOX). The alternative enzymes are peripheral, single-subunit oxido-reductases that do not pump protons. Thus, the oxidation of NADH via NDH2e-ubiquinone-AOX would not contribute to the proton-motive force. The futile oxidation of NADH may be prevented if either NDH2e or AOX bind to the classic complexes, channelling electrons. By oxymetry, it was observed that the electrons from complex I reached both cytochrome oxidase and AOX. In contrast, NDH2e-derived electrons were specifically channelled/directed to the cytochrome complexes. In addition, the presence of respiratory supercomplexes plus the interaction of NDH2e with these complexes was evaluated using blue native PAGE, clear native PAGE, in-gel activities, immunoblotting, mass spectrometry, and N-terminal sequencing. NDH2e (but not the redirected matrix NDH2i from a mutant strain, Δnubm) was detected in association with the cytochromic pathway; this interaction seems to be strong, as it was not disrupted by laurylmaltoside. The association of NDH2e to complex IV was also suggested when both enzymes coeluted from an ion exchange chromatography column. In Y. lipolytica mitochondria the cytochrome complexes probably associate into supercomplexes; those were assigned as follows: I–III 2, I–IV, I–III 2–IV 4, III 2–IV, III 2–IV 2, IV 2 and V 2. The molecular masses of all the complexes and putative supercomplexes detected in Y. lipolytica were estimated by comparison with the bovine mitochondrial complexes. To our knowledge, this is the first evidence of supercomplex formation in Y. lipolytica mitochondria and also, the first description of a specific association between an alternative NADH dehydrogenase and the classic cytochrome pathway.