Previous studies suggested that yeast ribosomal protein L26 was a candidate for the ribosomal subunit interface region. The present study used protein-protein cross-linking to identify neighboring proteins in intact 80S ribosomes of Saccharomyces cerevisiae. To facilitate identification of cross-linked pairs involving L26, 80S ribosomes were first treated with 5-iodoacetamidofluorescein to selectively label L26. Protein cross-links were produced with dithiobis[succinimidyl] propionate or N-succinimidyl-3-[2-pyridyldithio] propionate and analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing SDS. L26 was detected under UV and its cross-linked partner, detectable after staining with Coomassie blue, was located below the diagonal and was coincident with L26 on a single vertical axis. The identity of the partner was determined by its co-migration with 60S and 40S ribosomal protein markers on two-dimensional gels. Two protein pairs involving L26 and 40S subunit proteins were identified. The finding provided experimental evidence to support that L26 is located at the ribosomal subunit interface. A model that incorporates the present findings and the published data on intra-subunit protein pairs is proposed for the yeast 80S ribosome.
Cross-Linking Reagents Electrophoresis, Polyacrylamide Gel