Functional analysis of the apoptosis-inducing factor (AIF)
The apoptosis-inducing factor (AIF) is a phylogenetically old flavoprotein that is localised in mitochondria of healthy cells. It has been known to play an important role in the process of apoptosis, a genetically programmed cell suicide that removes supernumerous or potentially dangerous cells from a multicellular organism. Upon apoptosis induction, AIF translocates from the mitochondria via the cytosol to the nucleus, where it is involved in apoptosis-associated chromatinolysis. Here, I describe a hitherto unknown role of AIF in the functioning of the respiratory chain, the cell’s major source of energy. AIF-deficient murine embryonic stem cells were found to be highly dependent on glycolysis. This abnormal dependency was due to defective oxidative phosphorylation affecting in particular complex I and III of the respiratory chain. Similar observations were made in HeLa cells, in which the AIF expression had been downregulated by RNA interference. However, in the human cell line, reduced activity was found in complex I, but not in complex III. The respiratory chain defect was detectable not only at the functional but also at the protein level. All tested complex I subunits as well as the whole complex I were found to be less abundant in AIF-deficient cells than in their wild-type counterparts. This seemed to be a post-transcriptional phenomenon since the absence of AIF did not modify the levels of mRNA’s. Moreover, AIF does not seem to be a part of complex I or any other known respiratory chain complex, but retransfection with mitochondrially targeted AIF did cause re-expression of complex I and III subunits in AIF-KO cells. Since AIF has an oxidoreductase domain, it was speculated that AIF protected the respiratory chain complexes against ROS-induced damage, but treatment with several antioxidants could not restore the expression of complex I subunits in AIF-deficient cells. Therefore, AIF seems to play a role in the assembly or the maintenance of complex I and maybe other complexes. The existence of so-called supercomplexes, which comprise several respiratory chain complexes, could explain why the stability of more than one complex is affected. Harlequin mice, which exhibit only 20% of AIF expression due to a retroviral insertion in the Aif gene, were also found to have a complex I defect in different tissues including brain and retina but not heart and liver. That allowed for a re-interpretation of the harlequin phenotype, which is characterised by progressive neuronal and retinal degradation resembling symptoms of mitochondriopathies in humans. Harlequin mice can therefore serve as a mouse model to study complex I diseases. Moreover, it was shown that conditional knock-out mice (developed in Dr.Penninger’s laboratory) that did not express AIF in heart and muscle tissues and suffered from cardiomyopathy and muscle atrophy, had a respiratory chain defect in the affected tissues. In this animal model a complex I defect was also detected in the heart. It thus seems that AIF’s role in respiration is tissue-specific and also threshold-dependent. In a second part of this work, AIF’s role in apoptosis was further characterised by defining the interaction of AIF with nucleic acids. While the interaction of AIF with DNA had been described before, the binding of AIF to RNA was a new discovery. Recombinant AIF retained DNA fragments as well as RNA extracted from HeLa cells in a gel retention assay in a size and sequence independent manner. The direct interaction was also observed in electron microscopy experiments, where the presence of AIF induced the compaction of DNA and the formation of large aggregates of both DNA and RNA. Moreover, AIF showed a preferential binding to single-stranded over double-stranded DNA. Since different RNA molecules inhibited the retention of DNA by AIF, it was hypothesized that the nucleic acids bind to a similar or the same site of the protein. This hypothesis was further supported by the finding that two AIF mutants lacking positively charged amino acids, which had been described to mediate the AIF-DNA interaction, were less efficient in retaining both DNA and RNA in a horizontal agarose gel electrophoresis. Interestingly, the affinity for nucleic acids was enhanced by the co-factor NADP. While the role of the AIF-RNA interaction in vivo remains a conundrum, the AIF-DNA interaction plays an important role in chromatin condensation and, in certain cell death scenarios, large-scale DNA fragmentation.