Rbg1 associates with polyribosomes. (A) Whole-cell extracts from strain W303 were resolved by density sedimentation in 7 to 47% sucrose gradients. (B) Polyribosomes were disrupted by the addition of micrococcal nuclease (5 U/μl) prior to loading of extracts on gradients. The UV absorbance trace (254 nm) obtained during fractionation is shown with the positions of the 40S, 60S, 80S, and polyribosomes indicated. Fractions (numbered) were analyzed by immunoblotting with antibodies against Rbg1, Rbg2 and S2, a small ribosomal subunit protein, as indicated. L, 1/100 cell extract loaded on gradient.

Association of Gir2 with Gcn1 and Rbg1. (A) Copurification of Rbg1 and Gcn1 with GST-Gir2. GST and GST-Gir2 were affinity-purified with glutathione-Sepharose beads from yeast strains (H1511) expressing plasmid-borne GST alone or GST-Gir2 from a galactose-inducible promoter (pES128-9-1 and pJM3958, respectively). Aliquots of cellular extract, treated with or without RNase A, loaded onto the beads (input) or from the affinity-purified (bound) fractions, were analyzed by immunoblotting with antibodies to GST, Rbg1, or Gcn1, as indicated. (B) Cells expressing the GAL4 activation domain alone (pACT2) or fused to Gcn1(GIB)-AD [pACT2-GCN1(GIB)] and either the GAL4-binding domain alone (pAS2) or fused to Gcn2(GI), Gir2, Gir2N, or Gir2C [pAS-GCN2(GI), pAS-GIR2, pAS-GIR2N, and pAS-GIR2C, respectively] were analyzed for expression of HIS3 reporter gene by growth on SD−His−Trp−Leu plus 3-AT.

Gir2 partially cofractionates with translating ribosomes. Extracts from cells expressing Gir2-HA from the chromosome without (A) or with (B) the addition of micrococcal nuclease (5 U/μl) prior to fractionation on sucrose gradients. Immunoblots of indicated fractions reveal the positions of Gir2-HA and ribosomal protein S2. The positions of the 40S, 60S, and 80S monosomes and the polysomes are labeled. L, 1/100 extract loaded on gradient.

Modeling the interaction between translating ribosomes and Gcn2, Gcn1, Rbg1, and Gir2. (A) Gcn1 interacts with Gcn2 on the ribosome for detecting uncharged tRNAs under amino acid starvation. This leads to the stimulation of the Gcn2 kinase domain, subsequent phosphorylation of its substrate eIF-2α, and finally to the activation of the general amino acid control (see references 42 and 53). (B) On the ribosome, Gir2 binds simultaneously Rbg1 via its C-terminal domain, and Gcn1 binds via its N-terminal domain. In a manner analogous to the general amino control pathway described in panel A, the Rbg1 pathway may receive a signal from Gcn1 that is relayed through Gir2. It is possible that this signal alters the guanine nucleotide bound state of Rbg1 to elicit an as-yet-undefined biological response.

Rbg1 and Rbg2 are tripartite proteins, here illustrated as a diagram. Shaded regions represent regions of high similarity. The universally conserved G1 through G4 motifs of the guanine nucleotide binding domain are indicated by overlines. The C-terminal TGS domain is indicated. An amino acid reference scale is provided.

Interacting partners of Rbg1. A yeast two-hybrid screen identified Tma46, Gir2, Ygr250c, and Yap1 as potential interacting partners of Rbg1. The encoded protein fragments are depicted under a diagram of each full-length protein. Beginning and ending amino acids are indicated, as well as the number of clones for each fragment. Tma46 encodes a potential nuclear localization sequence and a zinc finger domain as indicated by the black and gray bars, respectively. Gir2 contains an N-terminal GI domain (black and white diamonds), as discussed in the text. Ygr250c possesses three potential RNA-binding motifs (diagonals), where indicated. The well-characterized Yap1 contains a basic-leucine zipper domain (black with dots).

Gir2 expression and amino acid control. (A) Overexpression of Gir2 leads to growth inhibition on 3-AT. Tenfold serial dilutions of cells harboring vector alone (p426-GAL1) or expressing GCN2(GI), GIR2, GIR2N, or GIR2C under the GAL1 promoter were spotted on plates containing either glucose or galactose, as indicated, in the presence of 10 mM 3-AT and incubated at 30°C. Similar strains that also express Gcn2 from a high-copy plasmid (hcGCN2; pAH15) were grown in the presence of galactose and 10 mM 3-AT. (B) Overexpression of hcGCN2 slightly perturbs the growth of starved cells. Tenfold serial dilutions of wild-type cells harboring hcGCN2 or vector alone in the absence (unstarved) or presence (starved) of 10 mM 3-AT. (C) Yeast strains (H1511) expressing plasmid-borne GST alone or GST-Gir2 and the isogenic gcn2Δ strain H2557 were grown to exponential phase and harvested, followed by immunoblot analysis with antibodies to eIF-2α and phosphorylated eIF-2α. Two different exposure times for phosphorylated eIF-2α are shown (two panels linked together by a gray bar on the right-hand side, with the upper panel showing short exposure and the lower panel showing long exposure). (D) Expression of GCN4-lacZ. Expression of GCN4 was determined in the strains indicated using a lacZ-based reported construct (p180), and the fold derepression was determined as the ratio of the wild-type values under nonstarvation conditions. For each strain, at least two independent transformants were assayed, in duplicate (gcn2Δ strain) or at least in triplicate, and the average values as well as the standard error values are shown in the graph.

Gir2 association with polysomes is partially dependent on Gcn1. Cell extracts from a wild-type strain (A), an rbg1Δ strain (B), or a gcn1Δ strain (C) were resolved by velocity sedimentation in sucrose gradients, and the positions of Gir2-HA and Rbg1 were detected by immunoblot analysis, as indicated. The positions of the 40S, 60S, and 80S monosomes and the polysomes are indicated. L, 1/100 extract loaded on gradient.