Interaction of Sso protein 2509 with aIF2. (A) Co-purification of Sso 2509 with aIF2 during outgrowth from prolonged starvation. Sso strain PH1-16(pMJ05-γ_His) was subjected to prolonged stationary phase and then reinoculated in Brock’s medium containing arabinose as described in ‘Materials and Methods’ section. Purified aIF2-α (lane 1), aIF2-ß (lane 2) and aIF2-γ (lane 3) were separated on a 12% SDS-polyacrylamide gel. The trimeric factor containing aIF2-γ_His was captured by Ni-NTA chromatography from PH1-16(pMJ05-γ_His) extracts obtained from cells subjected to prolonged starvation (lane 4) and 3 h after outgrowth from prolonged starvation (lane 5). The eluted proteins were separated on a 12% SDS polyacrylamide gel. A mock purification performed with extracts of strain PH1-16(pMJ05) obtained during outgrowth from prolonged stationary phase revealed nonspecific contaminant proteins (lane 6), which are indicated by stars. The bands corresponding to aIF2-α and aIF2-β co-purifying with aIF2-γ_His are indicated at the right. Protein Sso2509 (arrow) was found to associate predominantly with aIF2 during outgrowth. The positions of marker proteins are indicated at the left. The gels were stained with Coomassie brilliant blue. (B) Sso2509 removes aIF2 from the 5′-P₃-end of RNA. The immobilized 2508sh RNA was first saturated with aIF2. Unbound aIF2 present in the supernatant (S) was removed by several washing (W) steps. The presence and absence of aIF2 in the S (lane 1) and W (lane 2) fraction was confirmed by western blot analysis, respectively. The immobilized aIF2-2508sh complex was then either incubated with protein Sso2509 (lanes 3–5; + Sso2509) or with buffer (lanes 6–8; −Sso2509). The supernatant (S; lanes 3 and 6), the wash fraction (W; lanes 4 and 7) and the beads (B; lanes 5 and 8) were examined for the presence of aIF2(α,β,γ) by western blot analysis. The aIF2 subunits were detected using antibodies directed against either subunit protein.