Metabolism is the set of all chemical reactions that occur inside of cells. By providing all the building blocks that are required for sustaining a cellular state and cell proliferation, metabolism is at the core of cellular function. Therefore, in order to understand cellular function it is important to understand cellular metabolism. The cellular metabolic network comprises thousands of reactions even in the simplest of organisms. Due to the high complexity, a holistic approach is required to study and understand the interactions between different parts of metabolism giving rise to cellular phenotypes.In this thesis, a systems biology approach to study metabolism in yeast, mainly with a focus on Saccharomyces cerevisiae (baker’s yeast), was used. This approach consisted of combining proteomic analysis with constraint-based modeling to gain insights into different aspects of metabolism. First, the role of mitochondria in cellular metabolism throughout diauxic growth was evaluated, showing that mitochondria balance their role as a biosynthetic hub and center for energy generation depending on the mode of cellular metabolism. Next, the construction of a model of mitochondrial metabolism describing the essential mitochondrial processes of protein import and cofactor metabolism as well as proton motive force driving the generation of free energy (in the form of ATP) is described and evaluated. The model was used to investigate the dynamics in mitochondrial metabolism and the requirement of these processes.Second, the constraints placed on cellular metabolism arising from finite protein resources is investigated in two studies. The first study evaluates the effect of amino acid supplementation of the physiology and allocation of protein resources. This study showed that as the burden of producing amino acids is relieved, the cells can allocate more protein to the translation, which allows the cells to grow faster. In the second study, a quantitative comparison of four yeast species was performed to evaluate the underlying causes of overflow metabolism, which is the seemingly wasteful strategy of aerobic fermentation instead of using the more efficient respiratory pathway for glucose utilization. We showed that overflow metabolism in yeast is linked to adaptations in metabolism and protein translation This phenomenon is seen in cells ranging from bacteria to yeast and cancer cells, and the insights provided in our study could therefore be valuable in understanding the metabolism not only in yeast but in more complex systems
