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Steck, Regina (2010): Die Rolle des Proteins Fcj1 und der Untereinheiten Su e/Su g der F1FO-ATP-Synthase bei der Entstehung von Cristae und Crista Junctions in Mitochondrien. Dissertation, LMU München: Faculty of Biology
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Abstract

The crista junction is a tubular element of the mitochondrial inner membrane connecting inner boundary membrane and cristae membrane. The molecular basis of its formation was so far largely unknown. The lack of the mitochondrial inner membrane protein mitofilin results in the absence of crista junctions in HeLa-cells. Moreover, deletion of Fcj1, the orthologue of mitofilin in S. cerevisiae, leads to concentric stacks of cristae membranes and the virtual absence of crista junctions. The aim of the present work was to investigate the function of Fcj1 as well as the molecular basis of the formation of cristae and crista junctions in S. cerevisiae. Fcj1 undergoes homotypic interactions and is part of a stable oligomeric protein complex. Using immuno-EM, Fcj1 has been shown to be specifically enriched at crista junctions. All other proteins, which have been studied so far, such as subunits Su e and Su g of the F1FO-ATP-Synthase, are rather underrepresented in this region. Overexpression of Fcj1 increases the number of crista junctions, enlarges the crista junction diameter and leads to internal branching of cristae. Upon downregulation of Fcj1, the mitochondrial ultrastructure progressively changes reflecting the deletion phenotype more and more. Considering the fact that the decrease of the number of crista junctions occurs at an early time point, Fcj1 seems directly involved in the formation of crista junctions. In order to investigate if cristae form a detached compartment in the absence of crista junctions, the accessibility of the intracristal space for the protein-modifying substance AMS was examined. In fcj1 mitochondria the modification of cytochrome c was temporally delayed and incomplete in comparison to wild-type. Hence, metabolite exchange between intracristal space and cristae space seems impaired though not entirely impossible in the absence of crista junctions. This might be due to some extent of fusion and fission tracing back to protein or lipid exchange within the inner membrane of mitochondria. Cryo-EM tomograms revealed regular zipper-like arrangements of F1FO-ATP-Synthase particles. An inverse correlation of the amount of Fcj1 and the oligomeric state of F1FO-ATP-Synthase was observed upon biochemical analysis: in fcj1 mitochondria, F1FO-ATP-Synthase oligomers have a higher molecular weight and are more stable than in wild-type mitochondria, whereas in mitochondria of cells overexpressing Fcj1 they are smaller and less stable. Subunits Su e and Su g are essential for the stabilization of dimers and oligomers of the F1FO-ATP-Synthase. As the influence of Su e/Su g on the formation of crista junctions has not been investigated so far, the mitochondrial ultrastructure of both deletion strains was analyzed concerning this matter. Enlargement of crista junctions and internal branching of cristae similar to the overexpression phenotype of Fcj1 have been observed in both strains. Furthermore, crista tips are virtually absent. Apart from this functional interaction, Fcj1 and Su e/Su g have also been shown to interact genetically. Based on the gained insights on the roles of Fcj1 and Su e/Su g in cristae formation the following model is proposed: the modulation of the inner membrane of mitochondria is based on an antagonistic behavior of Fcj1 and Su e/Su g. Fcj1 introduces negative membrane curvatures, possibly by impairing the oligomerisation of the F1FO-ATP-Synthase. The presence of Su e/Su g, in contrast, promotes positive curvatures of cristae membranes by stabilizing dimers and oligomers of the F1FO-ATP-Synthase. Taken together, relative amounts of Fcj1 and Su e/Su g locally modulate the oligomeric state of the F1FO-ATP-Synthase and thereby enable the formation of crista junctions and crista tips in mitochondria.