Complexes containing rat liver 80s ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[/3,y-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with lZ5I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of M, 33000), P2, S6 and S23/ S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33 -L11 and LA33 -L12.During protein biosynthesis in eukaryotic cells, elongation factor 2 (EF-2) interacts with ribosomes in the presence of GTP and catalyzes the translocation of newly synthesized peptidyl-tRNA from the aminoacyl-tRNA binding site (the A site) of ribosomes to the peptidyl-tRNA site (the P site) [l -31. The functional roles of ribosomal proteins in the translocation are as yet unknown. To understand the molecular mechanisms of the translocation in mammalian ribosomes, it is important to identify proteins in the EF-2 binding site of ribosomes.Employing four kinds of cross-linking reagents and hydrogen peroxide, we identified 62 cross-linked protein pairs involving 36 protein species containing two acidic proteins of rat liver 60s subunits and have discussed these in relation to other functional data [4, 51. Concerning the 40s subunits, similar data were presented by Tolan and Traut [6]. Therefore, it is of interest that ribosomal proteins which interact with elongation factors have been determined.In the present experiments we investigated EF-2-binding sites in rat liver 80s ribosomes, employing 2-iminothiolane. Cross-linked proteins were analyzed by a modified method of diagonal SDS gel electrophoresis [7]. Seven large-subunit proteins and two small-subunit proteins were identified as the components cross-linked to EF-2. The mutual locations of these proteins in the 80s ribosomes are discussed.

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