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. 

The mitochondrial genome encodes only a handful of proteins, but methods to track their synthesis are highly limited. Saccharomyces cerevisiae is a model organism that offers possibilities to expand the classical systems to analyze mitochondrial translation. In this chapter, we present two approaches of monitoring mitochondrial protein synthesis. Labeling of mitochondrially translated products with radioactive amino acids can be performed either in intact cells or in isolated mitochondria. However, these classical methods have disadvantages that can affect cell physiology and hence are not suitable for all types of research questions. Some of these limitations can be overcome by the use of reporter genes that are inserted into yeast genetic screens mitochondrial DNA via biolistic transformation. These reporter genes can be used for yeast genetic screen and to monitor regulation and efficiency of mitochondrial translation with a variety of methods.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.