A systems biology approach to elucidating mechanisms underlying amino acid toxicities

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PROJECT SUMMARY Amino acids are an intrinsic part of protein biosynthesis, nucleotide production, and provide sources of carbon and nitrogen for the cell. The cell keeps amino acid (AA) levels balanced by increasing AA transporters on the cell surface, catabolizing AAs to form essential metabolites, protein translation, and storage of surplus AAs in the lysosome. An autophagic response is initiated when the cell is starved of AAs. Exposure to high concentrations of AAs is also problematic, but the mechanisms underlying that toxicity are not yet fully understood. AA levels are tightly regulated and when this regulation is disrupted, toxic intermediates can buildup and diseases such as phenylketonuria and cancer can occur. Previous research in our lab has studied amino acid toxicity through vacuole impairment, which is imprecise and cannot always provide a detailed understanding of the effects of single amino acids. To circumvent these issues, I have designed a yeast strain that overexpresses a mutated copy of Gap1, a high-capacity, low-specificity amino acid permease. Because of the two mutated Gap1 residues, this permease remains on the plasma membrane instead of being recycled to the vacuole in high AA conditions. This causes the tight AA regulation to be broken and initiate continuous AA uptake, which will allow us to study the effects of specific AAs on cellular processes. The aim of this project is to elucidate the role each AA plays in toxicity and cellular function using Saccharomyces cerevisiae as a model system. Aim 1 will identify transcriptional, metabolic, and organellar changes resulting from AA toxicity. Aim 2 aims to understand the cell’s capacity to buffer excess amino acids and adapt to their toxicity. Finally, the Aim 3 will characterize the mammalian plasma membrane transporter, L-type amino acid transporter (LAT1). LAT1 and its regulation are poorly characterized, and this aim will begin to fill this knowledge gap. Together, these proposed aims will help to uncover how AAs affect cellular function using the power of yeast genetics and it will begin to elucidate amino acid transporter regulation in mammalian cells.