Figure 2.

Subunit composition of eIF3, phosphorylation of eIF3 by cAMP-dependent kinase, and eIF3's binding to 40S subunits. (A) Coomassie staining of HeLa eIF3j+ and eIF3j- and autoradiography of eIF3j- and eIF3j+ ³²P-phosphorylated by cAMP-dependent kinase. eIF3 subunits are indicated. (B,C) Binding of ³²P-eIF3j+ and ³²P-eIF3j- to 40S subunits in the absence (B) and presence (C) of eIF1 and eIF1A, assayed by sucrose density gradient centrifugation and analyzed by optical density, Cerenkov counting (B,C) and gel electrophoresis (B, right). (D) 43S complex formation in the presence of 40S subunits, eIF2, [³⁵S]Met-, eIF1, eIF1A, and eIF3j+ or eIF3j- assayed by sucrose density gradient centrifugation and analyzed by optical density, scintillation counting, and gel electrophoresis (right). The optical density of eIF3j+/40S subunit complexes subjected to sucrose density gradient centrifugation is included for comparison of their mobility and that of 43S complexes. (E) Influence of rec-eIF3j subunit on binding of ³²P-eIF3j- to 40S subunits and binding of ³²P-eIF3j itself to 40S subunits assayed by sucrose density gradient centrifugation and analyzed by optical density and Cerenkov counting. (F) Influence of poly(U) RNA on binding of ³²P-eIF3j+ to 40S subunits assayed by sucrose density gradient centrifugation and analyzed by optical density and Cerenkov counting. Sedimentation was from right to left in all cases. Ribosomal complexes are indicated above appropriate peaks. Upper fractions from sucrose gradients have been omitted for clarity. (G) Comparison of the relative amounts of 40S subunits and ³²P-eIF3j+ in binary eIF3j+/40S subunit complexes and in 43S complexes separated by sucrose density gradient centrifugation and analyzed by fluorescent SYPRO staining (lanes 1,2) and autoradiography (lanes 3,4) after gel electrophoresis. (H) Presence of the eIF3j subunit from ³²P-eIF3j+ in different ribosomal complexes isolated by sucrose density gradient centrifugation.