Depletion of degron-tagged eIF4G1 in a mutant lacking eIF4G2 reduces, but does not abolish, translation initiation. (A) Degron-tagged eIF4G1 is undetectable after 8 h in nonpermissive conditions. Wildtype (WT) strain YAJ3 and tif4631-td tif4632Δ degron mutant YAJ41 were grown under permissive conditions to A600 of 0.3 and shifted to nonpermissive conditions for 4 h or 8 h. WCEs were prepared and subjected to Western blot analysis to monitor expression levels of eIF4G1 and Pab1 (examined as a loading control). (B) Polysome content is reduced in eIF4G-depleted cells. WT strain YAJ3 and the indicated degron mutants (YAJ41 and YAJ34) were grown under permissive conditions and shifted to nonpermissive conditions for 8 h. Following treatment with cycloheximide, WCEs were prepared and resolved by sedimentation through sucrose gradients. Gradient fractions were scanned at A254 to determine polysome/monosome ratios (P/M, mean+S.E.M., n = 4). The P/M ratio in the tif32-td prt1-td strain is an average from two independent gradients. The positions of the 40S and 60S ribosomal subunits, 80S monosome, light polysomes (LP), and heavy polysomes (HP) are indicated. (C) The rate of protein synthesis is reduced in eIF4G-depleted cells. The strains from (B) were cultured as described there and labeled with [35 S]-methionine. Acid-insoluble radioactivity, normalized for the A600 of the cells, was measured in aliquots of the cultures collected at the indicated times.