Abstract
Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5–5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.
Highlights
Eight different S. cerevisiae amino acid transporters were overexpressed by introducing their genes on multicopy plasmids under control of the constitutive ADH1 promoter (Figure 1)
Our study expands on the work of Risinger et al [41] who showed that all 20 proteinogenic amino acids, as well as the non-proteinogenic amino acids citrulline and ornithine, can be growth inhibitory to S. cerevisiae
We have shown that this effect can be mediated by various amino acid transporters and is not specific to Gap1
Summary
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