Ncl1 mediated metabolic rewiring critical during metabolic stress

Abstract

Nutritional limitation has been vastly studied, however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine upregulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation, events that are rescued by leucine supplementation. Through a comprehensive genetic screen we show that leucine mediated effect depends on a tRNA methyltransferase (Ncl1), absence of which decouples cell"s transcription and translation, inhibits the conversation of leucine to ketoisocaproate and leads to TCA cycle block. We therefore, propose a role of Ncl1 in regulating metabolic homeostasis through translational control.In this study, using Saccharomyces cerevisiae as a model, we used a combination of genetic, proteomic, transcriptomic and metabolic approach to understand cysteine induced systemic alteration. We show that cysteine inhibits protein translation and alters the amino acid metabolism, effects which are reversed by supplementation of leucine. We also show that a tRNA methyltransferase (NCL1) is involved in survival during cysteine stress, absence of which inhibited the conversation of leucine to ketoisocaproate (KIC), a necessary step for mitigating the effect of cysteine. Thus, this study not only uncovered the cellular insights during high levels cysteine, but also highlighted the novel role of NCL1 in regulating the metabolism during excess cysteine. Results Metabolic alterations due to excess amino acids induce growth defectGrowth screening of S cerevisiae (BY4741) in the presence of high concentrations of amino acids revealed that a few amino acids -cysteine, isoleucine, valine, tryptophan and phenylalanine inhibited growth (Fig. 1A). To test if the imbalance due to excess of an amino acid resulting in the growth inhibition could be abrogated in the presence of other amino acids, we did a comprehensive amino acid supplementation screening. We found that the amino acid, leucine could completely rescue the growth inhibition due to isoleucine, valine, tryptophan and phenylalanine but not cysteine, where the rescue was partial ( Fig 1B). The effect of leucine could be because the yeast strain used in this study, BY4741, is auxotrophic for leucine. To confirm this, we performed the same experiment using a prototrophic wild type strain, S288C, and found that except cysteine, none of the other amino acids inhibited growth ( Fig 1C). Further, the growth inhibitory effect of cysteine was lower compared to BY4741. This suggests that intracellular concentration of leucine might play a role in alleviating amino-acid mediated toxicity. This was further confirmed by deleting the gene invol...

Genome mining and UHPLC-MS/MS illuminate the specificity of secondary metabolite synthetic gene clusters in Bacillus subtilis NCD-2

“…During the microbial synthesis of secondary metabolites, such as lipopeptide, the relatively high energyconsuming process of protein synthesis takes priority [58]. Excessive energy consumption is not conducive to the normal growth of microbes, and, generally, microbes produce antibiotics in large amounts only when encountering pathogens or other stresses [59].…”