The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria

AbstractA DNA sequence homologous to non-proton-pumping NADH dehydrogenase genes was found in the genome of Neurospora crassa encoding a polypeptide of 577 amino acid residues, molecular mass of 64,656 Da, with a putative transmembrane domain. Analysis of fungal mitochondria fractionated with digitonin indicates that the protein is located at the outer face of the inner membrane of the organelle (external enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. Mitochondria from the resulting null-mutant nde2 are highly deficient in the oxidation of cytosolic NADH and NADPH. A triple mutant nde1/nde2/ndi1, lacking mitochondrial alternative NAD(P)H dehydrogenases, was obtained, indicating that these proteins are not essential in N. crassa. However, crosses between the nde2 mutant strain and complex I-deficient mutants yielded no viable double mutants. Transcription of the nde-2 gene, as well as of ndi-1 (internal enzyme), is repressed in the late exponential phase of fungal growth

Identification of the TYKY homologous subunit of complex I from Neurospora crassa

AbstractA polypeptide subunit of complex I from Neurospora crassa, homologous to bovine TYKY, was expressed in Escherichia coli, purified and used for the production of rabbit antiserum. The mature mitochondrial protein displays a molecular mass of 21280Da and results from cleavage of a presequence consisting of the first 34 N-terminal amino acids of the precursor. This protein was found closely associated with the peripheral arm of complex I

Biogénese do complexo I (NADH: coQ-Oxidoreductase) da cadeia respiratória de Neurospora crassa

Dissertação de Doutoramento em Ciências Biomédicas, área de especialização em Biologia Molecular, apresentada ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Port

Characterization of Apoptosis-Related Oxidoreductases from Neurospora crassa

The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins

Regulated Forms of Cell Death in Fungi

Cell death occurs in all domains of life. While some cells die in an uncontrolled way due to exposure to external cues, other cells die in a regulated manner as part of a genetically encoded developmental program. Like other eukaryotic species, fungi undergo programmed cell death (PCD) in response to various triggers. For example, exposure to external stress conditions can activate PCD pathways in fungi. Calcium redistribution between the extracellular space, the cytoplasm and intracellular storage organelles appears to be pivotal for this kind of cell death. PCD is also part of the fungal life cycle, in which it occurs during sexual and asexual reproduction, aging, and as part of development associated with infection in phytopathogenic fungi. Additionally, a fungal non-self-recognition mechanism termed heterokaryon incompatibility (HI) also involves PCD. Some of the molecular players mediating PCD during HI show remarkable similarities to major constituents involved in innate immunity in metazoans and plants. In this review we discuss recent research on fungal PCD mechanisms in comparison to more characterized mechanisms in metazoans. We highlight the role of PCD in fungi in response to exogenic compounds, fungal development and non-self-recognition processes and discuss identified intracellular signaling pathways and molecules that regulate fungal PCD

Mitochondrial type II NAD(P)H dehydrogenases in fungal cell death

During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(P)H dehydrogenases (also called alternative NAD(P)H dehydrogenases) are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(P)H dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(P)H dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF)-family

Programmed Cell Death in Neurospora crassa

Programmed cell death has been studied for decades in mammalian cells, but simpler organisms, including prokaryotes, plants, and fungi, also undergo regulated forms of cell death. We highlight the usefulness of the filamentous fungus Neurospora crassa as a model organism for the study of programmed cell death. In N. crassa, cell death can be triggered genetically due to hyphal fusion between individuals with different allelic specificities at het loci, in a process called “heterokaryon incompatibility.” Chemical induction of cell death can also be achieved upon exposure to death-inducing agents like staurosporine, phytosphingosine, or hydrogen peroxide. A summary of the recent advances made by our and other groups on the discovery of the mechanisms and mediators underlying the process of cell death in N. crassa is presented

The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria.

An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination