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. 2000 Apr;20(8):2706-17.
doi: 10.1128/MCB.20.8.2706-2717.2000.

Glucose limitation induces GCN4 translation by activation of Gcn2 protein kinase

R Yang  1 , S A Wek, R C Wek
Affiliations

Glucose limitation induces GCN4 translation by activation of Gcn2 protein kinase

R Yang et al. Mol Cell Biol. 2000 Apr.

Abstract

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) is a well-characterized mechanism regulating protein synthesis in response to environmental stresses. In the yeast Saccharomyces cerevisiae, starvation for amino acids induces phosphorylation of eIF-2alpha by Gcn2 protein kinase, leading to elevated translation of GCN4, a transcriptional activator of more than 50 genes. Uncharged tRNA that accumulates during amino acid limitation is proposed to activate Gcn2p by associating with Gcn2p sequences homologous to histidyl-tRNA synthetase (HisRS) enzymes. Given that eIF-2alpha phosphorylation in mammals is induced in response to both carbohydrate and amino acid limitations, we addressed whether activation of Gcn2p in yeast is also controlled by different nutrient deprivations. We found that starvation for glucose induces Gcn2p phosphorylation of eIF-2alpha and stimulates GCN4 translation. Induction of eIF-2alpha phosphorylation by Gcn2p during glucose limitation requires the function of the HisRS-related domain but is largely independent of the ribosome binding sequences of Gcn2p. Furthermore, Gcn20p, a factor required for Gcn2 protein kinase stimulation of GCN4 expression in response to amino acid starvation, is not essential for GCN4 translational control in response to limitation for carbohydrates. These results indicate there are differences between the mechanisms regulating Gcn2p activity in response to amino acid and carbohydrate deficiency. Gcn2p induction of GCN4 translation during carbohydrate limitation enhances storage of amino acids in the vacuoles and facilitates entry into exponential growth during a shift from low-glucose to high-glucose medium. Gcn2p function also contributes to maintenance of glycogen levels during prolonged glucose starvation, suggesting a linkage between amino acid control and glycogen metabolism.

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Figures

FIG. 1
FIG. 1
GCN4 expression is increased in response to starvation for either glucose or amino acids. (A) RY124 (GCN2) and RY133 (gcn2Δ) cells expressing GCN4-lacZ that included the four upstream ORFs in the 5′ noncoding region of GCN4 were cultured in minimal medium with 2% glucose (nonstarvation), 0.05% glucose (glucose starvation), 3% glycerol, or 3% ethanol. Cultures were collected, and Gcn4p-LacZ enzyme activity was measured in lysate preparations as detailed in Materials and Methods. (B) Four different yeast strains expressing GCN4-lacZ, including the four upstream ORFs, were cultured in SD medium (nonstarvation), minimal medium supplemented with 0.05% glucose (glucose starvation) or SD medium supplemented with 3-AT (amino acid starvation). In the case of the histidine auxotroph JC482, sulfometuron methyl was used to elicit starvation for amino acids instead of 3-AT (56). For each strain background, the fold increase of β-galactosidase activity during amino acid or glucose limitation compared with nonstarvation conditions is listed above the histograms.
FIG. 2
FIG. 2
Phosphorylation of eIF-2α is increased in yeast starved for glucose, amino acid, or purines. RY124 (GCN2) and RY133 (gcn2Δ) cells were cultured in minimal medium with 2% glucose (nonstarvation) or with 0.05% glucose (glucose starvation) or were grown in SD medium supplemented with 3-AT (amino acid starvation) or AzaA (purine starvation). Cell lysates were characterized by immunoblotting analysis using either a polyclonal antibody that specifically recognizes eIF-2α phosphorylated at serine-51 (A) or an antibody that recognizes both phosphorylated and nonphosphorylated forms of eIF-2α (B). The levels of phosphorylated eIF-2α in RY124 and RY133 cells cultured under each condition were measured by densitometry and presented as a histogram (C). Values are listed relative to that measured in RY124 grown under nonstarvation conditions.
FIG. 3
FIG. 3
The time course of increased phosphorylation of eIF-2α is coincident with induced GCN4 expression in response to glucose starvation. Wild-type RY124 cells were grown in SD medium to mid-logarithmic phase and then shifted to fresh minimal medium with 2% glucose (nonstarvation) or with 0.05% glucose (glucose starvation). Cells were incubated with shaking at 30°C, and aliquots of the cultures were analyzed at the indicated times for eIF-2α phosphorylation or for Gcn4p-LacZ enzyme activity. Time zero represents analysis of cells collected just prior to the shift of media. (A) Immunoblot analysis using a polyclonal antibody that specifically recognizes eIF-2α phosphorylated at serine-51. (B) Measurement of eIF-2α levels using a polyclonal antibody that recognizes both phosphorylated and nonphosphorylated forms of eIF-2α. (C) Gcn4p-LacZ enzyme activity in each culture sample.
FIG. 4
FIG. 4
Diagram of Gcn2 protein kinase mutants containing mutations in each of the Gcn2p domains. The box depicts the sequence of Gcn2 protein kinase, including the pseudo-kinase, protein kinase, HisRS-related, and ribosome association and dimerization domains. Gcn2p is 1,659 residues in length, 69 residues longer than previously reported (54). The location of mutations in gcn2-K628R, gcn2-m2, and gcn2-605 are indicated below the Gcn2p diagram. Sequences deleted in Gcn2p mutants are illustrated by brackets.
FIG. 5
FIG. 5
Impaired Gcn2p function delays growth following extended glucose starvation. RY124 (GCN2) and RY133 (gcn2Δ) cells were grown in minimal medium supplemented with 0.05% glucose for 22 h. Following this glucose-limited growth, cells were diluted into SD medium and incubated with shaking at 30°C. Cells were monitored for growth by measuring A600 and counting cell number. The gcn2Δ cells displayed a 2.5-h extension of the lag period prior to exponential growth in three independent experiments.
FIG. 6
FIG. 6
Measurement of vacuolar and cytoplasmic amino acid pools in wild-type and gcn2Δ cells cultured during nonstarvation and glucose starvation conditions. RY124 (GCN2) and RY133 (gcn2Δ) cells were grown in SD medium to mid-logarithmic phase and shifted to fresh minimal medium supplemented with either 2% glucose (nonstarvation) or 0.05% glucose (glucose starvation). Cell cultures were incubated with shaking at 30°C, and amino acid levels were measured in the cytoplasm (top) and vacuoles (bottom) by the ninhydrin method as described in Materials and Methods. Start point represents analysis of cells collected just prior to the shift of medium. Nonstarved cells were collected and analyzed after 4 h of growth in SD medium. Early and late glucose starvation represent analyses of cells collected after 6 and 20 h of incubation, respectively, in low-glucose medium. The inset represents the accumulation of vacuolar amino acid levels during early and late glucose starvation that exceeds the basal levels present at the start point.
FIG. 7
FIG. 7
Amino acids accumulate in the vacuole of gcn4Δ cells. (A) RY124 (GCN2), RY133 (gcn2Δ), and RY290 (gcn4Δ) cells were grown in SD medium to mid-logarithmic phase and shifted to minimal medium containing either 2% glucose (nonstarvation) or 0.05% glucose (glucose starvation). Cultures were incubated with shaking at 30°C, and vacuolar amino acid levels were measured as described in Materials and Methods. (B) As for panel A except that all 20 amino acids were added to the SD medium prior to the medium shift. Two independent experiments yielding similar results were carried out for each panel.
FIG. 8
FIG. 8
Glycogen levels are reduced in gcn2Δ mutants after extended starvation for glucose. RY124 (GCN2) and RY133 (gcn2Δ) were grown in SD medium to mid-logarithmic phase and then shifted to fresh minimal medium with 2% glucose (nonstarvation) or with 0.05% glucose (glucose starvation). Cell cultures were incubated with shaking at 30°C, and glycogen levels were measured in cells sampled at the indicated times. Start point represents analysis of cells just prior to the shift of medium.
FIG. 9
FIG. 9
Model for regulation of Gcn2 protein kinase and GCN4 translation during amino acid or glucose limitation. Uncharged tRNAs that accumulate during amino acid starvation are proposed to associate with the HisRS-related domain of Gcn2p, leading to a conformational change in the protein and activation of eIF-2α kinase activity (23, 52, 54, 56). Gcn1p and Gcn20p are required for elevated levels of eIF-2α phosphorylation by Gcn2p and are proposed to facilitate tRNA interaction with Gcn2p in the vicinity of the ribosome (31). Ribosomal association of Gcn2p is mediated by RNA-binding sequences in the carboxy terminus of Gcn2p, and this interaction is proposed to facilitate activation of the eIF-2α kinase during amino acid starvation (42, 60). Activation of Gcn2p during glucose limitation requires the function of the HisRS-related domain, suggesting uncharged tRNAs present during carbohydrate limitation signal activation of Gcn2p. Additional signals may also participate in the activation of this eIF-2α kinase during glucose limitation. While Gcn1p is required for regulation of Gcn2p during glucose starvation, Gcn20p is in part dispensable. It has been suggested that the EF3-like domain in Gcn1p facilitates delivery of uncharged tRNA to the HisRS-related domain of Gcn2p in the vicinity of the ribosomes (31). Gcn20p associates with Gcn1p and is proposed to enhance its regulatory function. The details of this enhancement are uncertain, but our results suggest that the role of Gcn20p is not simply to facilitate Gcn1p-mediated interaction of uncharged tRNA with Gcn2 protein kinase. Furthermore, Gcn2p sequences required for association of the protein kinase with ribosomes are not required for induction of GCN4 translation in response to glucose limitation. Elevated phosphorylation of eIF-2α by Gcn2 protein kinase during nutrient limitation reduces the exchange of eIF-2-GDP for eIF-2-GTP that is catalyzed by eIF-2B (24). After translation of upstream ORF1 in the 5′ leader of the GCN4 mRNA, the reduced eIF-2-GTP levels resulting from nutrient limitation are proposed to delay subsequent reinitiation of translation. This allows for the 40S ribosome devoid of eIF-2-GTP, as illustrated by the open circles, to scan through the inhibitory upstream ORF2, ORF3, and ORF4 located in the 5′ noncoding portion of the GCN4 mRNA. In the interval between ORF4 and the GCN4 coding sequences, scanning ribosomes associate with eIF-2–GTP and initiate translation at the GCN4 coding sequences.
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