FIG. 4.

In vivo interactions of eIF2Δ(K1K2K3). (A) Association with eIF2α and -γ determined by immunoprecipitation of eIF2β from a cell extract of strain 167-3C containing the SUI3 allele deleted of the sequences coding for the three lysine blocks present on the high-copy-number LEU2 plasmid YEp351 (pBE237) as well as the genes for eIF2α and -γ in the high-copy-number URA3 plasmid YEp352 (pBE188). Total yeast cell extract was incubated with antibodies raised against eIF2β (lane 1), eIF2α (lane 3), and eIF2γ (lane 4) and with preimmune serum (lane 2), adsorbed to agarose-protein A beads. The material bound to the beads was submitted to SDS-PAGE (10% polyacrylamide gel); the proteins were transferred to a nitrocellulose membrane and visualized by Western blotting with antibodies directed to eIF2β. (B) Association with ribosomal subunits. Fractions (1 through 13) obtained from a sucrose gradient of a cell extract from strain 167-3C/pBE237, prepared in the absence of cycloheximide, were subjected to Western blot analysis using antibodies directed to eIF2β. The peaks corresponding to the ribosomal subunits (40S and 60S) as well as the monosomes (80S) are indicated; E, cell extract before the gradient.