Fig. 7. Mapping the binding determinants in the PK domain for the HisRS-N region and evidence that the GCN2^c-E803V mutation impairs interaction between the PK and C-term domains in vivo. (A) Fusion proteins containing the truncated GCN2 PK domain (residues 720–999) or its deletion derivatives (depicted by gray rectangular boxes) and the B42 transcription activation domain were expressed from the plasmids listed on the left in transformants of yeast strain EGY48 coexpressing LexA–HisRS-N(970–1156) (from pHQ689) or LexA–C-term(1498–1659) (from pHQ311). Dashed lines represent the amino acids deleted from the PK domain, and the GCN2 residue numbers above the lines indicate the first and last residues deleted in each construct. The resulting transformants were tested for growth on SGal/Raf + Ura medium, indicative of a two-hybrid interaction that activates expression of the lexAop-LEU2 reporter resident in the strains, as previously described (Qiu et al., 1998). +, –/+ and – indicate strong, weak and no growth on the medium lacking leucine, respectively. (B) In vitro binding of [³⁵S]HisRS-N (aa 970–1156) synthesized in vitro to GST fusion proteins containing different segments of the PK domain. The upper panel displays the amount of [³⁵S]HisRS-N added to the reactions (1/10 Input) or bound to the GST or GST–PK fusion proteins as indicated above the panel. 1× and 2× represent two different relative concentrations of GST–PK fusion proteins used in the binding reactions.The lower panel shows the GST and GST–PK fusion proteins bound to the glutathione–Sepharose beads in the binding reactions stained with Coomassie Brilliant Blue. The full-length GST–PK fusion proteins are indicated by white dots next to the appropriate bands. (C) The GCN2^c-E803V mutation impairs two-hybrid interaction between the PK and C-term domains in vivo. LexA fusion proteins containing truncated GCN2 PK(720–999) (encoded by pHQ433), C-term(1498–1659) (encoded by pHQ311) or HisRS-N(970–1156) (encoded by pHQ689) were expressed in transformants of EGY48 expressing B42 fusion proteins containing PK(720–999) (from pHQ428) or PK(720–999)/E803V (from pHQ823). The resulting transformants were tested for growth on SGal/Raf + Ura medium (lacking leucine), which is indicative of a two-hybrid interaction that activates expression of the lexAop-LEU2 reporter.