Abstract
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.
Highlights
Mitochondria are regarded as the powerhouse of the cell producing the high levels of ATP required for life and death [1,2]
N. crassa depicts three apoptosis inducing factor (AIF)-like proteins The genes coding for putative oxidoreductases belonging to the protein family of apoptosis-inducing factors AIF, AMID and AMID2 were identified in a BLAST search of the genomic database of the Neurospora Sequencing Project at the Whitehead Institute/MIT Center for Genome Research using the previously characterized S. cerevisiae AIF protein sequence as query [29]
All three genes belong to linkage group VII, AIF is composed of 2 exons encoding a polypeptide of 612 amino acids, AMID is composed of five exons encoding a polypeptide of 434 aminoacids and AMID2 is composed of five exons encoding a polypeptide of 447 aminoacids
Summary
Mitochondria are regarded as the powerhouse of the cell producing the high levels of ATP required for life and death [1,2]. Mitochondria energy production is accomplished through a series of complexes present in the inner mitochondrial membrane that carry along oxidative phosphorylation and are known as OXPHOS complexes, of which type I NADH:ubiquinone oxidoreductase or complex I is the largest and most complex one [7,8]. Complex I, composed of up to 45 subunits in mammals, couples electron transfer from NADH to ubiquinone with proton pumping across the inner mitochondrial membrane contributing a great deal to the proton motive force that will be used for ATP synthesis [9,10]. Many organisms are known to contain highly branched mitochondrial respiratory chains encompassing type II NAD(P)H dehydrogenases that bypass complex I transferring electrons to ubiquinone in a rotenone-insensitive manner [11,12]. Alternative dehydrogenases, described in bacteria, protozoa, plants and fungi, have been proposed to be energy conservation bypasses and to provide plasticity under adverse environmental conditions, their precise physiological relevance remains unclear [14,15,12]
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