Untersuchung der submitochondrialen Verteilung von Oxa1 und Mba1 in Saccharomyces cerevisiae

Abstract

The mitochondria of eucaryotic cells are involved in several cellular processes and fulfill a multitude of fundamental tasks, such as the production of energy via the process of oxidative phosphorylation. They exhibit a high structural complexity. The outer mitochondrial membrane encloses the entire organelle and therefore delimits it against the cytoplasm. The highly infolded inner mitochondrial membrane is organized into two contiguous but morphologically distinct domains: the “cristae membrane” and the “inner boundary membrane” which faces the outer mitochondrial membrane. Previous studies provided evidence for a functional compartmentalization of the inner mitochondrial membrane because of the heterogeneous distribution of several mitochondrial proteins. In the course of these studies, the protein Oxa1 of the baker’s yeast Saccharomyces cerevisiae was examined, which plays a key role not only in the insertion of nuclear encoded proteins but also of proteins encoded in the mitochondrial genome. A detailed analysis concerning the distribution of Oxa1 revealed it’s preferential localization in the inner boundary membrane when yeast cells are grown with a fermentable carbon source. However, this heterogeneous distribution changes dynamically depending on the physiological needs of the cell. Thus, in yeast cells grown with a non-fermentable carbon source, Oxa1 is enriched in the cristae membrane. A preferential localization in the cristae membrane can also be observed for Oxa1 when the evolutionary conserved amino acid tryptophan at position 128 is substituted with phenylalanine. Furthermore, the substitution mutation oxa1-W128F resulted in a decrease in the size of Oxa1 containing complexes leading to the question whether a correlation exists between the complex sizes and the submitochondrial localization of Oxa1. Therefore, the sizes of the Oxa1 containing complexes were analyzed depending on the available carbon source. In the course of this work, it has been shown that the sizes of Oxa1 containing complexes, which are located in the inner boundary membrane, do not differ from the complexes that are enriched in the cristae membrane. Accordingly, a correlation between the sizes of Oxa1 containing complexes and the submitochondrial localization of Oxa1 could not be demonstrated, and the shift in Oxa1 distribution must therefore be regulated by another mechanism. The analysis of the mutated variant Oxa1-W128F showed a reduction in the complex size in comparison to Oxa1, like it has been described previously. Further, the observed sizes of Oxa1-W128F containing complexes are in accordance to the size of an Oxa1 dimer as well as an Oxa1 tetramer, and the decrease in Oxa1 complex size can be traced back to an impairment in homooligomerization. In conjunction with the fact that no further interaction partner besides Oxa1 itself could be identified, these data suggest that Oxa1 might function as a homooligomeric complex under fermentative as well as respiratory growth conditions, and that the dynamic behaviour of Oxa1 distribution could be attributed to transient interactions with substrates. For S. cerevisiae, it has been well established that in addition to Oxa1, further mitochondrial proteins are part of the protein insertion machinery located in the inner mitochondrial membrane, including Pnt1 and Mba1. To date, no conclusive data concerning the distribution of Pnt1 and Mba1 within the inner membrane were available. In this thesis, it could be demonstrated that both Pnt1 and Mba1 are heterogeneously distributed with an enrichment in the inner boundary membrane under fermentative growth conditions. In the case of Pnt1, the subjection of the yeast cells to respiratory growth conditions resulted in a change of the preferential localization from the inner boundary membrane to the cristae membrane, like it has been reported for Oxa1. In contrast to Oxa1 and Pnt1, the protein Mba1 remained enriched in the inner boundary membrane. Therefore, the present study could show that the heterogeneous distribution of Pnt1 displays a dynamic which is dependent on the physiological conditions, whereas the heterogeneous distribution of Mba1 is static. Based on the diverse localization behaviour of Mba1 in comparison to Oxa1 and Pnt1, a more detailed investigation of the submitochondrial localization of Mba1 was performed. The analysis of the causes leading to the static heterogeneous distribution of Mba1 on a molecular level, revealed that already the deletion of five amino acids (mba1 1-273) as well as the substitution of a single amino acid (mba1-I272A) of the C-terminal part of Mba1 results in a drastic alteration of Mba1 distribution. The study of the consequences of the gene mutations on Mba1 function showed that the deletion mutation causes a decreased respiration competency of the yeast cells which can be attributed to an impairment in the assembly of a super complex of the respiratory chain (2 x complexIII / 2 x complexIV). Therefore, this thesis provides evidence that the static heterogeneous distribution of Mba1 within the inner mitochondrial membrane is crucial for Mba1 function. It is possible that the binding of a currently unknown interaction partner determines the location of Mba1 function.