FIGURE 7.

Int6/eIF3e promotes de novo protein synthesis. A, cultures of haploid S. pombe strains WY764 (WT, row 1) and KAY252 (int6Δ, row 2) were spotted onto EMM + adenine medium with (+) or without (-) 4 mm 3AT, as described in the legend to Fig. 1A, and incubated at 30 °C for 6 and 5 days, respectively. B, Atf1 abundance was monitored using strains in A, with affinity-purified anti-Atf1 antibodies (21), exactly as described in the legend to Fig. 5, A and B, but using EMM + adenine medium. The graph indicates relative abundances for Atf1 after correction by tubulin abundance with S.D. in lines (n = 3). C, WY764 (WT, lanes 1–4) and KAY252 (int6Δ, lanes 5–8) were cultured in the presence of 3AT for up to 3 h, as indicated. Prior to harvesting, cultures were pulse-labeled with [³⁵S]methionine, and the total proteins in the harvested cells were analyzed by SDS-PAGE. Radiolabeled proteins were visualized by phosphorimaging (bottom panel). To ensure normalization of total proteins analyzed in the SDS-PAGE, the top panel shows Coomassie staining of this gel. Lane M, size standards. D, quantitation of radiolabeled proteins in response to 3AT treatment from panel C is presented with the value in lane 1 (no 3AT treatment) indicated as 100%. Averages from three independent experiments are shown with S.D. in lines. Column numbers corresponds to lane numbers in C.