Phosphorylation of eIF2α is induced in response to starvation for different amino acids. Isogenic yeast strains were subjected to amino acid starvation by treating a prototrophic strain WY798 with 3-AT (A) or introducing auxotrophic strains WY900 (trp1) and WY909 (his3) into SC medium depleted of tryptophan (SC-Trp) or histidine (SC-His), respectively, for up to 60 min (B). WY795 (GCN2 leu2) and WY796 (gcn2 leu2) were also cultured in SC medium devoid of leucine (SC-Leu) for up to 120 min (C). Cells cultured in SC medium containing a full complement of amino acids are represented as zero time in each of the panels. Equal amounts of protein lysates were separated by SDS-PAGE, and the levels of eIF2α phosphorylated specifically at serine 51 (eIF2α∼P) or total eIF2α were measured by immunoblot analyses using antibodies specific to each.

Genome-wide analysis of tRNA charging. A, the charging of each chromosomal-encoded tRNA in S. cerevisiae was measured by a method that involved selective labeling of charged tRNA with Cy3 or Cy5 fluorophore and hybridization to tRNA microarrays containing complementary probes to each tRNA. Yeast cells were starved for amino acids, and total tRNA was extracted under conditions that retain aminoacylated tRNAs. The total RNA samples were divided into two portions. One part was treated with periodate, which oxidizes the 3′-acceptor stem of uncharged tRNA, whereas the other part was exposed only to buffer solution. The tRNAs in both parts were deacylated and then ligated to a fluorescent-tagged oligonucleotide, which contains a stable stem-loop structure and a portion complementary to the 3′-CCA sequence that is conserved among all tRNAs. Both of the samples were labeled with Cy3 or Cy5 fluorophore and were combined and hybridized to microarrays that contained probes for each chromosomal-encoded tRNA. The first microarray used Cy5-labeled charged tRNA and Cy3-labeled total tRNA followed by a second microarray, which utilized Cy3-labeled charged tRNA and Cy5-labeled total tRNA. B, representative spots from scanned fluorescent images of tRNA^His (His-tRNA) hybridized to a complementary probe in the microarrays. The tRNA preparations were from cells treated with 3-AT for 60 min (60min) or no starvation (0). Green intensity measurements indicate decreased tRNA^His charging, and yellow indicates no difference in tRNA aminoacylation. C, relative tRNA charging levels are presented as the ratio of charging of each tRNA listed prepared from cells cultured in the presence of 3-AT for 15 min (+3AT, 15′) or 60 min (+3AT, 60′) when compared with non-starved cells. The x axis indicates each of the different tRNAs, separated into hydrophobic, small, charged, and polar groups. In the y axis, the value of 1.0 represents equal tRNA charging in the presence of 3-AT when compared with absence of 3-AT (non-starved control). Values less than 1.0 indicate reduced tRNA charging in response to 3-AT treatment, whereas values greater than 1.0 represent enhanced charging of the indicated tRNA. Heat map representations of the genome-wide tRNA charging in response to 3-AT and other subsequent stress conditions are represented in supplemental Fig. S1. Error bars indicate S.E. D, comparison of the tRNA charging levels measurements by the microarray method when compared with that measured by Northern blot analysis of acid-denaturing gels. The numbers indicate the charging of specific tRNAs charging in the prototroph strain treated with 3-AT or the auxotroph strains starved for histidine, leucine, or tryptophan, as indicated by the legend. +3AT, 60′, cells cultured in the presence of 3-AT for 60 min ; −His, 60′, cells cultured in SC medium devoid of His for 60 min; −Leu, 60′, cells cultured in SC medium devoid of Leu for 60 min; −Trp, 15′, cells cultured in SC medium devoid of Trp for 15 min; −Trp, 60′, cells cultured in SC medium devoid of Trp for 60 min. E, Northern blot analysis of acid-denaturing gels measuring the charging of tRNA^His in strain WY798 treated with 3-AT for 1 h (+3AT, 60′) or with no amino acid starvation (No 3AT). The panels illustrate an autoradiogram representing hybridization of a radiolabeled probe complementary to charged (slower migrating band) and uncharged (faster migrating band) tRNA^His. As a control, the tRNA^His was deacylated prior to the Northern analysis (+) and when compared with the tRNA preparations, which were not subjected to deacylation in vitro prior to the Northern analysis (−).

Changes in tRNA charging in response to starvation for histidine. The auxotrophic strain WY909 (his3) was cultured in SC medium depleted of histidine for up to 60 min, and the levels of charged tRNAs were measured using the microarray method. A, scanned fluorescent images of tRNA^His (His-tRNA) and tRNA^Asp (Asp-tRNA) hybridized to complementary probe spots in the microarray. The tRNA preparations were from cells starved for histidine for 60 min (60min) or no starvation (0). Green specifies decreased tRNA charging, and yellow indicates no change in the aminoacylation levels of the tRNA. B, relative tRNA charging levels are presented as the charging ratio of each tRNA prepared from cells cultured in SC medium devoid of histidine for 15 min (−His, 15′) or 60 min (−His, 60′) when compared with cells grown in SC medium containing all amino acids. The x axis indicates each of the different tRNAs, and the value of 1.0 in the y axis represents that tRNA charging in the histidine starvation condition equals that in the non-starved control. Reduced tRNA charging during histidine starvation is less than 1.0, whereas values greater than 1.0 represent increased tRNA charging. Error bars indicate S.E.

Multiple leucyl-tRNA isoacceptors are deacylated in response to leucine starvation. The auxotrophic strain WY795 (leu2) was cultured in SC medium depleted of leucine for up to 60 min, and tRNA charging was measured using the microarray method. A, scanned fluorescent images of tRNA^His (His-tRNA) and tRNA^Leu(UAA) (Leu(UAA)) hybridized to the microarrays. The tRNA preparations are from cells starved for leucine for 60 min (60min) or no starvation (0). Green and yellow specify decreased tRNA charging and no change, respectively. B, relative levels of tRNA charging are presented as the charging ratio of each tRNA prepared from the strain cultured in SC medium devoid of leucine for 15 min (−Leu, 15′) or 60 min (−Leu, 60′) when compared with cells grown in SC medium containing all amino acids. The x axis includes each of the chromosomal-encoded tRNAs, and the value of 1.0 in the y axis indicates that the tRNA charging in cells starved for leucine is equal to the non-starved control. Values lower than 1.0 indicate reduced tRNA charging, whereas values greater than 1.0 represent tRNA charging that is greater in response to leucine starvation. Error bars indicate S.E.

Multiple tRNAs are deacylated in response to tryptophan starvation. The auxotrophic strain WY900 (trp1) was cultured in SC medium depleted of tryptophan for up to 60 min, and tRNA charging was measured by the microarray method. A, scanned fluorescent images of tRNA^His (His-tRNA), tRNA^Trp (Trp-tRNA), and tRNA^Asp (Asp-tRNA) hybridized to the complementary probes in the microarrays. The tRNA preparations were from cells starved for tryptophan for 60 min (60min) or no starvation (0). Green indicates decreased tRNA charging, and yellow represents no change. B, the relative levels of tRNA charging are presented as the charging ratio of each tRNA prepared from the strain cultured in SC medium devoid of tryptophan for 15 min (−Trp, 15′) or 60 min (−Trp, 60′) when compared with cells grown in SC medium containing all amino acids. The x axis lists each tRNA. The value of 1.0 in the y axis indicates that the tRNA charging in the tryptophan starvation condition is equal to cells grown in non-starved conditions. Values less than 1.0 indicate reduced tRNA charging, whereas values greater than 1.0 represent increased tRNA charging in response to tryptophan starvation. Error bars indicate S.E.

Amino acid composition in yeast cells subjected to histidine starvation by treatment with 3-AT or histidine depletion of an auxotrophic strain. A, yeast strain WY798 was cultured in SC medium devoid of histidine containing 3-AT for 15 min (+3AT 15min) or 60 min (+3AT 60min). Alternatively, non-starved cells (Control) were grown in the SC medium without 3-AT. Amino acid profiles in whole cell lysates were measured by the Waters Pico-Tag method. B, strain WY909 (his3) was cultured in SC medium depleted of histidine for 15 min (−His 15min) or 60 min (−His 60min). Additionally, cells were cultured in SC medium containing all amino acids (Control). Amino acid compositions were measured in whole cell lysates. Amino acid measurements with significant changes during these starvation conditions when compared with control are illustrated with an asterisk where the asterisk indicates p < 0.05. The levels of amino acids that did not significantly change in response to 3-AT treatment are presented in supplemental Fig. S2. Error bars indicate S.E.

Composition of amino acids in an auxotrophic strain starved for leucine. Strain WY795 (leu2) was cultured in SC medium devoid of leucine for 15 min (−Leu 15min) or 60 min (−Leu 60min) or in SC medium containing all amino acids (Control). The amino acid levels were determined in the whole cell lysates using the Waters Pico-Tag method. Amino acid measurements with significant changes in response to leucine deprivation when compared with control are illustrated with an asterisk where the asterisk indicates p < 0.05. The levels of amino acids that did not significantly change in response to leucine starvation are presented in supplemental Fig. S2. Error bars indicate S.E.

Gcn2p phosphorylation of eIF2α is enhanced during treatment with 1 m NaCl. Prototrophic cells WY798 and an isogenic gcn2 strain (WY799) were cultured in SC medium containing 1 m NaCl for up to 120 min. Cells grown in SC and not subject to the high salt stress are indicated by the zero times. Equal amounts of protein lysates were separated by SDS-PAGE. Levels of eIF2α phosphorylated at serine 51 (eIF2α∼P) or total eIF2α were measured by immunoblot analyses.

Increased uncharged tRNA levels in response to treatment with high salt treatment. The prototrophic strain WY798 was cultured in SC medium containing 1 m NaCl for up to 60 min, and the charging of tRNA genome-wide was measured by the microarray method. A, scanned fluorescent images of tRNA_i^Met (Met-i) and tRNA^Cys (Cys-tRNA) hybridized to the complementary probes in the microarrays. The tRNA preparations were from cells treated with 1 m NaCl for 15 min (15 min) or no stress (0). Green indicates low tRNA charging, and yellow represents no change in the charging of tRNA. B, the relative levels of tRNA charging are presented as the ratio of each charged tRNA prepared from the strain cultured in high salt conditions for 15 min (+NaCl, 15′) or 60 min (+NaCl, 60′) when compared with cells grown in the absence of stress. The x axis lists each tRNA, and the y axis represents changes in tRNA charging between stressed and non-stressed cultures. Values less than 1.0 indicate reduced tRNA charging, whereas values above 1.0 denote increased tRNA charging in response to the 1 m NaCl treatment. Error bars indicate S.E. C, Northern blot analysis of acid-denaturing gels measuring the charging of tRNA_i^Met in strain WY798 treated with 1 m NaCl for 15 min (+NaCl, 15′), 60 min (+NaCl, 60′), or no stress (No NaCl). Autoradiograms represent the charged (slower migrating band) and uncharged (faster migrating band) tRNA_i^Met. As a control, the tRNA_i^Met was deacylated prior to the Northern analysis (+) and when compared with those not subjected to deacylation in vitro prior to the Northern analysis (−).