Significance of mitochondrial ultrastructure for bioenergetics

Abstract: Mitochondria are the site where most of the energy from food is converted into adenosine triphosphate (ATP). This process is taking place at the inner membrane (IM) of mitochondria, and is called oxidative phosphorylation, and results in the establishment of a proton motive force (pmf). The proton motive force is a combination of a proton difference over the mitochondrial IM and a charge difference. The ATP is then synthesized by the ATP synthase, which is utilizing the pmf for this process. The IM of mitochondria has many invaginations, which are called cristae. The enzymes of the respiratory chain are mainly located at the flat sheet part, while the ATP synthase is located at the rim of the cristae. The hypothesis arises whether the cristae membrane would serve as a proton sink for the ATP synthase, due to the curved shape of the cristae. We aimed at answering this hypothesis by attaching a pH-sensitive green fluorescent protein (pHluorin2) at different locations within the mitochondria. The study revealed that there is no substantial pH difference across the IM of yeast mitochondria and that the cristae are not functioning as a proton sink, but rather its main function is to provide an optimal environment, for coupled enzymatic activity. The second project investigated the importance of the mobile electron carrier; cytochrome c (cyt c) of its ability to freely diffuse along with the IM. Cyt c is the electron carrier between the bc1 complex and cytochrome c oxidase of the respiratory chain. It is also involved in programmed cell death (apoptosis) of higher eukaryotes, where its release from mitochondria initiates apoptosis. As its role in yeast apoptosis is not entirely clear, we created a yeast strain where cyt c was tethered to the IM, in a background strain that was devoid of the mobile cyt c. Interestingly, the level of apoptosis was higher in the yeast strain with the non-mobile cyt c, which indicated that cyt c release in yeast is not a necessary step to initiate apoptosis. The strain with the IM tethered cyt c had also higher levels of reactive oxygen species (ROS), shorter life span, alterations of the mitochondrial network in comparison to the wild type strain. Despite not showing any major alterations in the respiratory chain, the mutant yeast strain had elevated oxygen consumption, indicating a compensatory mechanism, which could have caused the elevated ROS levels which ultimately induced apoptosis. Maintaining a steady level of ATP is crucial for the cell, and one such mechanism in higher eukaryotes is the creatine phosphate shuttle system, by the enzyme; creatine kinase. Creatine kinase is catalyzing the phosphorylation of creatine in mitochondria, and the phosphocreatine is transported out to the cytosol, where the cytoplasmic isoform of the enzyme is regenerating ATP from the phosphocreatine. A yeast strain was created to express the mitochondrial creatine kinase, which could serve as a strategy in industrially relevant yeast strains, to circumvent ATP levels to drop during the production processes. To gain an understanding of the importance of cyt c diffusion, its relevance for the respiratory chain, the yeast strain from the second project was modified so that it was fused to the bc1 complex. The strain showed a functional respiratory chain, and further work will provide insights into the diffusion of the respiratory complexes and their interaction with the IM.