Ncl1-mediated metabolic rewiring critical during metabolic stress

(A) Cells were treated with cysteine for 6 and 12 h, and then iTRAQ-based relative quantification of proteins was performed. The figure represents the directionality of the expression of proteins involved in amino acid metabolism during 6 and 12 h of cysteine treatment. Left-side and right-side boxes for each protein denote its expression at 6 and 12 h of cysteine treatment, respectively. Red, green, and beige colored boxes represent the proteins down-regulated, up-regulated, and not changed, respectively, because of cysteine treatment. However, white colored box represents the proteins not detected in the proteomics experiment. (B) Heat map represents the relative expression of proteins (with respect to untreated condition) involved in amino acid metabolism during cysteine treatment and their status during co-treatment of leucine and cysteine. (C) Heat map represents the log fold change of different amino acids during cysteine (Cys) treatment and combination of leucine and cysteine (Leu + Cys). Statistical significance was determined by t test (n = 5, and n = 2 for Gln, Asn, and Trp, respectively). In the first column, the amino acids significantly altered by cysteine are labeled by “*” (P < 0.05); however, if the levels of these amino acids are significantly reverted by co-treatment of leucine and cysteine, then in the second column they are labeled by “*” (P < 0.05) or otherwise “ns” (nonsignificant). (D)35S Methionine incorporation kinetics in the presence of different concentrations (1 and 2 mM) of cysteine for different time points. In the upper panel, the upper blot is for autoradiogram, which signifies S³⁵-Met incorporation in proteins, and the lower blot represents Coomassie stain of the same proteins, which represents equal loading of proteins. The lower panel is the quantification of 35S incorporation with respect to untreated cells. “**” indicates P < 0.01 (t test). Error bar represents SEM (n = 2). (E) Polysome profiling representing monosome and polysome fractions in the presence and absence of cysteine treatment and during co-treatment of leucine and cysteine.

(A) Whole genome screen in the presence of 1 mM cysteine. The upper most panel represents the schematic of the genome-wide screen. The middle panel represents scatter plot between the logarithmic ratio of growth in the presence and absence of cysteine versus growth in basal media (SC media), for each deletion strain. In this panel, the deletion strains, which show growth sensitivity and resistance for cysteine, are symbolized with red and blue dots, respectively. Strains that were very slow growing and show growth advantage with cysteine are denoted by green dots. The lower most panel represents the validation of the cysteine-sensitive strains. Strains that show growth sensitivity toward high levels of cysteine in first round of whole genome screen were again grown in the presence of cysteine and growth was monitored and compared with wild-type (Wt) strain. Three biological replicates were performed, and average percentage growth of each strain with respect to Wt was calculated. Deletion strains whose growth was significantly different from Wt (P-value ≤ 0.05) (statistical significance was determined by unpaired t test), and grows at least 20% less than Wt, were considered to be sensitive for cysteine and are denoted by red dots. (B) Screening to identify genes required by leucine for alleviating cysteine-induced toxicity. Cysteine-sensitive deletion strains were grown with and without higher concentration of cysteine (1 mM), leucine (5 mM), or combination of leucine and cysteine. The figure represents the scatter plot between percentage growth during cysteine treatment versus percentage growth during co- treatment with leucine and cysteine, with respect to wild type’s growth during cysteine treatment. Deletion strains in the left quadrant, which are denoted by red dots, represent the strains that are sensitive to cysteine but are not recovered by leucine.

(A) Percentage growth inhibition of wild-type (Wt) and ncl1Δ strain, in the presence of cysteine and combination of leucine and cysteine. (B) Box plot represents the relative expression of proteins during co-treatment of leucine and cysteine in Wt and ncl1Δ, for proteins that were up-regulated by cysteine in Wt. Each condition represents the log2-fold change of different proteins (n = 32), which is an average of three biological replicates, and statistical significance was analyzed using paired nonparametric method. (C) Spearman rank correlation between relative expression of mRNA (obtained from RNA-seq) and protein levels (from iTRAQ-based relative proteomics) in Wt (upper panel) and ncl1Δ (lower panel). (D) The bar graph represents the biological pathways enriched from proteins whose expression was reverted during co-treatment of leucine and cysteine in Wt, but not to the similar extent in Δncl1. Classification was done using Database for Annotation, Visualization & Integrated Discovery (DAVID), and pathways with P-value ≤ 0.05 are plotted in the figure.

(A) Relative amino acid (AA) levels in ncl1Δ strain during Cys treatment and combination of Leu and Cys treatment were measured and were analyzed by calculating the correlation between these two groups. (B) Bar graph represents the relative levels of leucine in Wt and ncl1Δ cells, during leucine and combination of leucine and cysteine, normalized with respect to levels of leucine in untreated Wt strain. Error bar represents SD (n = 2). Statistical significance was determined by unpaired t test. “**” indicates P < 0.01. (C) Relative expression of BAT1 in Wt and ncl1Δ during cysteine treatment, normalized with respect to its expression in cysteine-treated Wt. Error bar represents SD (n = 3). Statistical significance was determined by paired t test “*” indicates P < 0.05. (D) KIC levels were measured using MRM-based LC–MS technique. The figure represents the overlapped peak intensity of a transition of KIC (121/101 m/z), measured from Wt and ncl1Δ cells during co-treatment of leucine and cysteine. KIC was also measured in both these cells during co-treatment of KIC and cysteine. (E) Cells were co-treated with cysteine (1 mM) and different concentrations of leucine or KIC, and then growth was measured after 12 h. The graph represents the growth inhibition of Wt and ncl1Δ strain because of cysteine in the basal media and during its co-treatment with varying concentration of KIC and leucine. (F) Growth of cells in the presence of toxic amino acids (Ile, Val, Phe, and Trp) and during their respective co-treatment with KIC. (G) TCA intermediates were measured by MRM-based LC–MS technique. The bar plot represents their relative levels during cysteine treatment and its combination with KIC or leucine in ncl1Δ strain. Error bar represents SD (n = 3).