Crosslinking of eukaryotic initiation factor eIF3 to the 40S ribosomal subunit from rabbit reticulocytes.
Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.
D000818:Animals; D003432:Cross-Linking Reagents; D005057:Eukaryotic Cells; D039621:Eukaryotic Initiation Factor-3; D007096:Imidoesters; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D003432:Cross-Linking Reagents; D039621:Eukaryotic Initiation Factor-3; D007096:Imidoesters; D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins; C005905:methyl 4-mercaptobutyrimidate