Identification of Initiation Factors and Ribosome-Associated Phosphoproteins by Two-Dimensional Polyacrylamide Gel Electrophoresis

Abstract

A 2-dimensional polyacrylamide gel electrophoresis procedure was used to identify initiation factors rapidly in the high-salt-wash fraction from [rabbit] reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which was used to maintain [.gamma.-32P]ATP and [.gamma.-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex were identified using a combination of the 2 procedures. The salt-wash fraction contains 8 major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins comigrate with 2 of the 3 subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf to 40-S subunits. Three phosphorylated proteins co-migrate with eIF-3, the factor facilitating entry of Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. The eIF-4B, 1 of the proteins involved in binding mRNA to 40-S subunits, is also phosphorylated. The remainder of the phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and were not identified further. Of the 6 phosphoproteins associated with the salt-washed ribosomes, 2 comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits and the other is an acidicprotein.