Growth inhibition by amino acids inSaccharomyces cerevisiae

Abstract

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced down-regulation 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.

MCH4is a multicopy suppressor of glycine toxicity inSaccharomyces cerevisiae

“…The mechanism of amino acid toxicity in the GAP1 UR strain is still not understood. Several mechanisms such as dissipation of the proton gradient, induction of the GCN pathway, and mischarging of tRNAs were considered in previous reports (Risinger et al, 2006;Ruiz et al, 2017). Based on our result in the mutant with disrupted HOG signaling pathway, we can add osmotic shock to this list.…”

“…While it is most pronounced under synthetic conditions (overexpression of unregulated Gap1p), it is likely present in natural context as evidenced by numerous reports of amino acid secretion under conditions of perturbed metabolism (Casalone et al, 1997;Feller et al, 1999;Hess et al, 2006;Lewis and Phaff, 1964;Delgado et al, 1982;Velasco et al, 2005), peptide uptake (Melnykov, 2016), or simply upon entry into the stationary phase of growth (Paczia et al, 2012). Among several phenotypes sensitive to amino acids (Ljungdahl et al, 1992;Risinger et al, 2006;Ruiz et al, 2017), we chose the GAP1 UR strain for our genetic selection experiment as the one with a broad spectrum of sensitivity.…”

“…Amino acid toxicity has been reported in budding yeast under conditions of unregulated uptake through the general amino acid permease (Gap1p) (Risinger et al, 2006) and several other broad specificity transporters (Ruiz et al, 2017). Regulation of GAP1 is understood in greatest detail; it is regulated transcriptionally by the quality of the nitrogen source and posttranslationally by intracellular amino acid concentrations.…”

“…Generally, as Gap1p is a transporter of broad specificity, all natural amino acids except alanine inhibit growth of the GAP1 UR strain to some degree (Risinger et al, 2006). Similarly, overexpression of AGP1, BAP2, CAN1, DIP5, GNP1, LYP1, PUT4, or TAT2 from a multicopy plasmid resulted in amino acid sensitivity (Ruiz et al, 2017). In each case, several amino acids were found to inhibit growth, and the spectrum of inhibitory amino acids was specific to the transporter reflecting known and newly discovered transport specificities.…”

Lipid Production from Amino Acid Wastes by the Oleaginous Yeast Rhodosporidium toruloides

“…Pro at high initial concentrations may exert more osmotic stress [5,27,32], leading to inhibitory effects. For the Pro-28 media, when the culture time increased to 180 h, the cell mass and lipid content were 8.7 g/L and 37.3%, respectively, and there was 22.4 g/L Pro leftover.…”