Fig. 6. Arg2259 in GCN1 is essential for GCN2 binding and GCN1 regulatory function. (A) GST–GCN1[2052–2428] directly interacts with His₆–GCN2[1–598] in vitro, dependent on Arg2259. The indicated GST–GCN1 fusions or GST alone encoded by plasmids (from left to right) pES164-2A, ES123-B1 or pGEX-6p-1 were expressed in E.coli and immobilized on glutathione–Sepharose beads, then incubated with E.coli extract containing His₆–GCN2[1–598] encoded by plasmid pES171-III-1. Proteins bound to the beads were identified by immunoblot analysis, as described in Figure 4 using anti-His₆ antibodies. GST proteins were visualized by Coomassie staining. (B) Arg2259 in GCN1 is essential for GCN1–GCN2 interaction in vivo. Co-immunoprecipitation assays were performed as described in Figure 3A using transformants of gcn1Δ strain H2256 harboring plasmid-borne gcn1-R2259A (pES174-3-2), GCN1 (p2367) or vector alone (gcn1Δ,pRS316), as indicated at the top of the panel. Immunoblots were probed for GCN1, GCN2 and GCN20. (C) Arg2259 is essential for GCN1 function. Serial dilutions of gcn1Δ strain H2556 harboring the indicated gcn1 alleles on plasmid (from top to bottom) p2367, pES161-1-2, pES174-3-2, pES179-1-2 or vector pRS316 alone (gcn1Δ) were spotted on plates containing 3AT as indicated and incubated at 30°C. (D) gcn1-R2259A confers a dominant Gcn^– phenotype. GCN1 strain H1511 containing plasmid-borne gcn1 alleles as shown (plasmids as in C), or gcn1Δ strain H2556 harboring vector alone, were subjected to a dominance test as described in Figure 5A. (E) GCN2 overexpression suppresses the Gcn^– phenotype of gcn1-R2259A. gcn1Δ strain H2556 harboring gcn1 alleles as indicated (plasmids as in C) and high copy GCN2 plasmid pAH15 or vector YEp13 alone were studied for growth on medium containing 3AT, as in (D).