42 Regulation of the Mitochondrial Oxaloacetate OAC1

Mentor: Zachary Wilson (Biochemistry, University of Utah)

Mitochondria are organelles critical for the generation of metabolic energy in eukaryotic cells. To perform this function, mitochondria must constantly 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. These proteins are essential to the regulation of several mitochondrial processes including the citric acid cycle, ATP production, and many others. Despite their importance, little is known about how mitochondrial metabolite carriers are regulated within the mitochondrial inner membrane.

To investigate the function and regulation of mitochondrial metabolite carriers, we examined one carrier, the mitochondrial oxaloacetate carrier (Oac1), in the Baker’s yeast Saccharomyces cerevisiae. We demonstrated that the Oac1 protein remains functional when fused with small epitope-tags at the N- or C-terminus. In particular, we examined the Oac1 protein in yeast strains tagged with either a Myc or HA epitope tag. By using these two different epitope tags, we unexpectedly discovered growth conditions in which the Oac1-HA protein became specifically depleted.

Upon transferring yeast cells from nutrient-rich media into media lacking specific essential nutrients, it was discovered that Oac1-HA levels decreased when switched into media lacking uracil or methionine. Similar to the nutrient-rich condition, however, Oac1-HA protein levels remained steady when the cells were moved into media lacking leucine. Conversely, we observed that the Oac1-Myc protein levels remained steady in all media conditions. A follow-up experiment was then conducted to determine whether the Oac1-HA protein was becoming stabilized in the minus leucine condition or if the protein was instead being synthesized more rapidly. To examine these possibilities, yeast strains containing Oac1-HA and Oac1-Myc were grown in nutrient-rich media then briefly incubated in either nutrient-rich media or media lacking leucine before treatment with cycloheximide, a protein translation inhibitor. We observed that Oac1-HA protein levels decreased in both media conditions after protein synthesis was arrested, while Oac1-Myc protein levels remained steady over 3 hours. This result indicated that Oac1-HA was being rapidly degraded once protein synthesis was arrested, suggesting that the HA tag destabilizes the Oac1 protein. After discovering the media conditions in which the Oac1-HA protein became destabilized, several other HA-tagged mitochondrial proteins were examined to determine if they would provide similar results. Using truncated forms of Oac1 and tagging several other mitochondrial proteins, we showed that the HA-epitope tag generally destabilizes proteins localized within mitochondria.

The observed differences in yeast strains containing Oac1-HA versus Oac1-Myc indicate that HA-tagged Oac1 is more susceptible to being degraded when placed in certain altered metabolic growth conditions. After this discovery, we found evidence in the literature that others have seen a similar instability in HA-tagged proteins located within the cytoplasm. Future experiments will explore if the HA-epitope tag can be used to control mitochondrial protein levels if paired to a regulatable promoter. Because the HA tag appears to be inherently unstable in certain conditions, we also plan to continue experimenting with the more stable Oac1-Myc yeast strains in order to find conditions that specifically alter Oac1 protein levels.