Mitochondria are semi-autonomous organelles that harbor one of the main cellular processes – energy conversion. Nutrients available in the environment are taken up by cells and transformed into metabolites that can be used for synthesis of ATP, the energy currency of the cell. In mitochondria, ATP synthesis depends on oxidative phosphorylation (OXPHOS), a metabolic pathway that engages multimeric protein complexes embedded in the mitochondrial inner membrane. Despite that most of their subunits are translated in the cytoplasm and transported into mitochondria, core subunits of the OXPHOS complexes are encoded in mitochondrial DNA (mtDNA). Therefore, this handful of proteins encoded in mtDNA and synthesized by the mitochondrial gene expression system is crucial for energy conversion. Underlying mechanisms regulating their synthesis are of interest for better understanding of mitochondrial gene expression and ATP synthesis. 

The functionality of the OXPHOS complexes is dependent on the coordinated expression and assembly of their subunits, yet ATP production in cells is regulated by the cellular metabolism. In Saccharomyces cerevisiae the preferable metabolic state is fermentation, during which ATP is synthesized by the glycolysis pathway in the cytoplasm. Upon exhaustion of glucose, cells are required to adjust their metabolism to use another carbon source. By switching from fermentation to respiration, cells engage OXPHOS for more efficient ATP production in a low glucose environment. The increased ATP production requires adjustment of the whole cellular metabolism. These adjustments have been extensively studied on nuclear gene expression and whole cellular levels, but little is known about mitochondrial adaptations to the shift between these two metabolic states. Mitochondrial translation as well as cellular metabolism are therefore important aspects regulating ATP synthesis. However, studies on these processes are limited by available experimental techniques. 

In my thesis, I addressed this problem by developing new methods to follow mitochondrial gene expression. Employing S. cerevisiae as a model organism, it was possible to introduce into mitochondrial DNA reporter genes that code for green fluorescent protein (GFP) or a luminescent protein (nanoluciferase). Moreover, I showed that nanoluciferase can be used for studies on mitochondrial adaptation to metabolic changes in cells. Measuring nanoluciferase activity, we could observe rapid and reversible changes in mitochondrial functions that were induced by a switch of available nutrients in the growth media. These changes in nanoluciferase activity suggested an existence of a signaling pathway between cytosol and mitochondria that can regulate mitochondrial homeostasis and quickly tune its functions in response to cellular metabolic needs independently of mitochondrial gene expression. 

In summary, this work presents a new and versatile approach to modify mitochondrial DNA to study mitochondrial gene expression and homeostasis. Mitochondrially encoded reporters broaden the available toolkit to follow mitochondrial protein synthesis. Moreover, nanoluciferase activity was shown to follow the metabolic state of the cells and gave more insights into regulation of cellular energy conversion.