(494.3) Elucidating the cellular mechanism of methionine toxicity

Poster Board Number: A156

Emily Pitsch (University of Utah), Adam Hughes (University of Utah)

Cells possess several mechanisms to maintain amino acid homeostasis including regulation of amino acid permease trafficking, protein synthesis, metabolic reactions, and storage within the lysosome or vacuole. With aging and in certain diseases these processes are impaired, leading to a toxic accumulation of amino acids. While it is well-established that excess amino acids are problematic for cells, it is poorly understood how individual amino acids drive cellular dysfunction and how cells combat toxic levels of these metabolites. To begin to elucidate cellular mechanisms of amino acid toxicity, we focused our analysis on methionine, because of its unique role in translation, metabolism, and as a precursor for the methyl donor, s-adenosylmethionine. Additionally, methionine restriction has been shown to extend lifespan and delay progression or improve disease phenotypes, suggesting that methionine itself can be problematic to organisms. 

To study methionine toxicity, we first identified yeast strains that were sensitive to the amino acid. We genetically modulated pathways cells use to traffic two amino acid permeases – the high-affinity methionine permease (Mup1) and the general amino acid permease (Gap1). These permeases are targeted for removal from the plasma membrane by ubiquitination of N-terminal lysine residues, which is facilitated by an α-arrestin protein. Mup1 internalization was inhibited by knocking out the α-arrestin, LDB19, and Gap1 internalization was blocked usingGAP1K9R,K16R. Compared to wildtype, these mutants were greatly sensitized to exogenous methionine, measured by growth inhibition, highlighting the importance of modulating methionine levels to maintain cellular health. To explore mechanisms of methionine toxicity, we conducted RNA-sequencing and found that excess methionine leads to an upregulation of genes under transcriptional regulation of Hsf1 and Gcn4. These transcription factors are activated in response to disruptions in protein homeostasis and tRNA charging, respectively. In addition to Hsf1 activation, we observed aggregation of heat shock proteins, Hsp104 and Hsp42, via fluorescent microscopy. Genetic depletion of Hsf1 using a tet-repressible allele led to diminished growth on methionine compared to control cells, suggesting there is an increased requirement for Hsf1 when cells are exposed to excess methionine. Finally, we found a decrease in total protein when cells are exposed to excess methionine. This could be explained by the activation of Gcn4 which is activated in response to alternations in tRNA homeostasis and would reduce translation rates. 

The reasons for methionine toxicity are currently unknown, but our data suggests that excess methionine drives proteotoxicity and may perturb tRNA charging homeostasis. This work provides us with preliminary evidence for the mechanisms through which methionine imparts toxicity and how cells respond to excess levels of this amino acid.

Support or Funding Information

Funding: NIH, R01 AG061376