Studies on the external dehydrogenases
Studies on the external dehydrogenases of N. crassa have shown that the NDE1 protein is a Ca2+-dependent external NADPH dehydrogenase, while NDE2 is characterized as a dehydrogenase oxidizing both cytosolic substrates (external NADH and/or NADPH) (Carneiro et al., 2004, Melo et al., 2001). All three dehydrogenases of N. crassa (NDI1 and NDE1,2) are not essential proteins for the function of the fungus as a triple mutant lacking these three enzymes has been obtained (Carneiro et al. 2004). According to the authors, an expression profile of the genes encoding these three dehydrogenases revealed that the nde2 and ndi1 genes are highly downregulated, probably in a coordinated manner, in the late exponential phase of N. crassa growth, in tachykinin receptor to the nde1 gene. Similar to NDI1, the NDE2 dehydrogenase may contribute to spore germination as nde2 mutants exhibit a much slower germination rate compared to the wild-type N. crassa. The failure to obtain a double mutant (nde2cI mutant) lacking both NDE2 and Complex I unexpectedly suggested that NDI1 is unable to compensate for the lack of Complex I in the N. crassa mitochondria (Carneiro et al. 2004). Additional studies have surprisingly implied that NDE2 complements Complex I activity. This observation indicates some ways of exchanging the matrix/cytosolic NADH that are present in N. crassa. Based on experiments analyzing the Ca2+ influence on the mutant strains, it has been proposed that Ca2+ somehow allows for the cytosolic NADH to pass the inner mitochondrial membrane (Carneiro et al., 2004, Melo et al., 2001). Whether the observed effects are due to Ca2+ regulation of the permeability transition pore (PTP) still remains unknown. An NDE3 of a predicted molecular mass of ∼55kDa has been localized both in the mitochondria and in the cytosol of N. crassa and its sequence shows no typical features of a mitochondrial cleavable targeting sequence (Carneiro et al. 2007). This enzyme has been proven to be anchored in the inner mitochondrial membrane and face the intermembrane space. Similar to NDE2, NDE3 oxidizes both NADH and NADPH. In contrast to the nde2cI mutant, the nde3cI mutant has been obtained. Despite the fact that NDE2 has been considered to be the primary external NAD(P)H dehydrogenase, the contribution of NDE3 to the cytosolic oxidation of NAD(P)H has been shown to be greater than previously expected. In the nde3 mutant, the nde2 expression increases significantly. However, in the wild-type strain, the upregulation of the nde3 transcript in contrast to the significant downregulation of nde2 and ndi1 has been observed from the early to late exponential phase of growth. These data indicate that the activities of particular dehydrogenases could possibly be partially compensated by others during the different stages of N. crassa development. These compensations could enable the fungus to adapt to changing environmental conditions, and the dual location of NDE3 may serve as a type of sensing mechanism (Carneiro et al. 2007). It has been also investigated whether the alternative NAD(P)H dehydrogenases may be a significant source of ROS generation in mitochondria with a branched respiratory chain (Carneiro et al. 2012). Using paraquat, it was found that N. crassa strains devoid of one or more alternative dehydrogenases exhibit an increased tolerance to this redox cycling agent compared to the wild-type strain. In particular, the double mutant lacking NDE1 and NDE2 was the most resistant to the reducing agent, although it did not withstand H2O2 and heat shock stresses. In addition, decreased levels of ROS have been observed in the nde1nde2 mutant strain, and among alternative dehydrogenases, NDE2 has been suggested to play a major role in ROS generation in the N. crassa mitochondria (Carneiro et al. 2012). Yarrowia lipolyticaIn the obligate aerobic filamentous fungus Y. lipolytica, only one gene encoding an alternative dehydrogenase has been identified and designated YLNDH2 (Kerscher et al. 1999). The YLNDH2 enzyme is located externally in the inner mitochondrial membrane. Deletion of the gene encoding the enzyme has no influence on the Y. lipolytica mutant viability. Therefore, other mechanisms responsible for cytosolic NADH oxidation must also be present in the Y. lipolytica mitochondria. The phylogenetic analysis of known NADH type II dehydrogenases revealed that a common ancestor of fungal alternative NADH:UQ oxidoreductases originally had an external orientation and that the internal dehydrogenase form, such as that of S. cerevisiae, emerged from divergent evolution following an early gene duplication event (Kerscher et al. 1999). Further studies showed that YLNDH2 is engaged in supercomplex formation with Complexes III and IV, depending on the cell growth phase (Guerrero-Castillo et al., 2009, Guerrero-Castillo et al., 2012). In mitochondria from high energy-requiring cells in the logarithmic growth phase, most YLNDH2 protein was associated with cytochrome c oxidase (Complex IV), and electrons from NADH were channeled to the cytochrome pathway (Guerrero-Castillo et al. 2012). In contrast, in the low energy-requiring, late stationary-growth phase, Complex IV concentration decreased, and the cells overexpressed YLNDH2. Thus, a large fraction of this enzyme was found in a non-associated form. The AOX-sustained pathway was activated at the same time, and ROS production also decreased. These association/dissociation processes of YLNDH2 to Complex IV have been proposed to be a switch that channels electrons either to the energy-conserving cytochrome pathway or the energy-dissipating AOX-sustained pathway. The latter entirely nonproton-pumping electron pathway decreases the reduction level of respiratory chain electron carriers, thus preventing the overproduction of ROS. In Y. lypolytica mitochondria, as in S. cerevisiae mitochondria, it has been suggested that the formation of supercomplexes promotes the channeling of respiratory substrates and electrons, the sequestration of ROS, and the stabilization of labile, multisubunit complexes (Guerrero-Castillo et al., 2009, Guerrero-Castillo et al., 2012).