Abstract
Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.
Highlights
The effects of amino acid availability on the regulation of protein synthesis are believed to be mediated predominantly through the mechanistic target of rapamycin complex 1 pathway, which appears to be especially sensitive to leucine availability[1,2], and through regulation of the phosphorylation status of the alpha subunit of eukaryotic initiation factor 2[3]
To evaluate the effects of single amino acid deprivation on cellular 4EBP1 and eukaryotic initiation factor 2 (eIF2) phosphorylation and protein synthesis in HEK293T cells, we chose a time point of 12 h after the switch from complete to amino acid-deficient medium. This time period allowed both sufficient depletion of the intracellular essential amino acid pool of the limiting amino acid to impact mRNA translation as well as time for cells to recover from the integrated stress response that is often observed in response to a change in culture medium
Deprivation of arginine, histidine, leucine or methionine each resulted in an ~25% decrease in cell growth/proliferation as measured by total protein content of the cultured cells compared to cells grown in sufficient medium
Summary
The effects of amino acid availability on the regulation of protein synthesis are believed to be mediated predominantly through the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which appears to be especially sensitive to leucine availability[1,2], and through regulation of the phosphorylation status of the alpha subunit of eukaryotic initiation factor 2 (eIF2)[3]. Single essential amino acid deprivation can affect hepatic polysome profiles[27,28,29,30], rates of hepatic protein synthesis[30,31], and the phosphorylation status of 4EBP127,30–32 and eIF229–31,33–36. We further explore effects of single essential amino acid deprivation on 4EBP1 and eIF2 phosphorylation status and protein synthesis
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