| Description |
Mitochondria are organelles known for their role in many critical cellular processes including the production of metabolic energy in eukaryotic cells. In order to produce this energy, mitochondria continually transport metabolites across the impermeable mitochondrial inner membrane. The transport of metabolites into and out of mitochondria is controlled by a family of proteins called mitochondrial metabolite carriers. One carrier known as the mitochondrial oxaloacetate carrier (Oac1) in the Baker's yeast Saccharomyces cerevisiae was investigated in this thesis. It was demonstrated that the Oac1 protein remains functional when fused with small epitope tags at its N- or C-terminus. However, we noticed that when Oac1 is C-terminally fused to a hemagglutinin (HA) epitope tag it becomes destabilized and is rapidly degraded. By HA-tagging several other mitochondrial proteins and truncated forms of Oac1, it was shown that the HA-epitope tag generally destabilizes proteins localized within mitochondria. Here, we attempted to functionalize this HA-epitope tag by combining this destabilizing element with an RNA element that can be induced to form a translation-inhibiting RNA aptamer upon binding the antibiotic tetracycline (TC). By combining these two elements, we show that inhibiting Oac1-HA translation by TC addition leads to the rapid and specific turnover of the Oac1-HA protein. Thus, pairing a yeast-specific HA-epitope tag to a regulatable TC-binding RNA aptamer creates a tool that can be used to induce specific protein degradation in mitochondria. Future experiments may explore the induced degradation of various mitochondrial proteins in order to study their function and regulation. |
