62 Characterizing Metabolic and Transcriptional Changes Caused by Amino Acid Toxicity

Faculty Mentor: Kylie Jacobs (Biochemistry, University of Utah)

Amino acids play an essential role in cell metabolism as the building block of proteins. It is known that amino acid deficiency in cells causes the cell to initiate autophagy, however it is not well understood how they react to elevated amino acid conditions. Research suggests that elevated levels of amino acids are deleterious to the cell and organismal health, however there is little understanding of how this amino acid toxicity affects metabolic and transcriptional systems in the cell. Certain metabolic disorders such as diabetes and citrullinemia have been associated with elevated levels of amino acids; however many of these metabolic disorders do not have effective treatments beyond dietary restrictions which is why it is important that we understand how amino acid affects metabolic systems. The aim of this research is to investigate how amino acid toxicity affects the metabolic and transcriptional cell systems of budding yeast in order to create new therapies for metabolic disorders. In order to identify proteins involved in evading amino acid toxicity, we generated a library of suppressor strains that resist toxicity. Suppressors are cells that developed a mutation which allows them to grow in conditions that would otherwise be lethal such as high concentrations of amino acids. We generated this suppressor library by plating cells susceptible to amino acids onto high concentrations of each amino acid and selecting colonies that were able to survive those conditions. We then tested all of these strains on high concentrations of amino acids to determine their ability to resist toxic amino acid concentrations. Those that were able to resist the toxic effects of some amino acids, but not all amino acids were selected for further genetic, metabolic, and transcriptional analysis to identify pathways involved in ablating the toxicity of specific amino acids. From whole genome sequencing we were able to identify frameshift mutations in three genes: super histidine resistance 3 (Shr3), BI3, and Gap1. The Shr3 gene is required for the incorporation of amino acid permeases into the plasma membrane through ER chaperoning, a deletion of these gene results in amino acid permeases being stuck in the ER and unable to reach the plasma membrane. BI3 is a mitochondrial mRNA maturase that forms a complex with Mrs1 to mediate Cytochrome B (COB) splicing, a mutation in this gene results in the shortening of BI4 and the improper splicing of COB rendering the cell non-respiratory. Gap1 is an amino acid permease which is normally shuttled to and from the plasma membrane depending on the cell’s necessity for amino acids. However, we have developed a Gap1 mutant that allows it to stay on the plasma membrane in order to sensitize the cell to amino acids. These Gap1 mutants in the suppressors are gaining a frameshift mutation which renders our mutant nonfunctional. In the future we plan to recreate these mutations in wild type yeast cells to test how amino acid toxicity affects organelle morphology, its effect on metabolic pathways, and observe localization of amino acid permeases. As this is an ongoing project, we do not yet fully understand how amino acid toxicity causes changes in metabolic and transcriptional functions, but we hope that
further testing will allow us to shed light on this mystery.