The External Alternative NAD(P)H Dehydrogenase NDE3 Is Localized both in the Mitochondria and in the Cytoplasm of Neurospora crassa

The filamentous fungus Neurospora crassa has a branched respiratory chain. Several alternative dehydrogenases, aside from the canonical complex I enzyme, are involved in the oxidation of NAD(P)H substrates. Based on homology searches in the fungal genome, we have tentatively identified one of these proteins. The corresponding gene was inactivated by the generation of repeat-induced point mutations and a null-mutant strain was isolated. This mutant is deficient in the oxidation of cytosolic NADH, and to a lesser extent NADPH. Thus, a fourth mitochondrial alternative NAD(P)H dehydrogenase, named NDE3, was recognized in N. crassa. Interestingly, a combination of Western blot analysis of cell fractions and the in vivo detection of the protein fused to the green fluorescent protein revealed that it is also located in the fungal cytoplasm. In contrast to the other NAD(P)H dehydrogenases, expression of the nde-3 gene is up-regulated in the late exponential growth phase of N. crassa. The absence of the protein results in an up-regulation of the nde-2 transcript in this phase of growth, suggesting that the proteins are important in specific stages of fungal development. The identification of the proteins responsible for the entry point of electrons from NAD(P)H into the respiratory chain of N. crassa is likely completed.