Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor

Abstract

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, was identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) was purified from high salt (0.88 M KCl) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity was measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity was recovered from most of the particles that were resolved by zonal centrifugation of the total native subunits obtained from the postmicrosomal fraction; the highest concentration of IF-3 was associated with native 40S subunits. The purified dissociation factor IF-3 was composed of about 10 polypeptides and the MW was from 500,000-700,000, on the basis of glycerol and CeCl gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes were dissociated by IF-3, a product was formed which was dependent on the concentration of the protein factor and had the characteristics of a 40S-IF-3 complex; centrifugation of the complex on sucrose and CeCl gradients suggests that the complex consists of 1 equivalent of each of the 2 components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appeared to interact with 60S subunits but multiple complexes were formed, some with more than 1 IF-3 equivalent/60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it did not affect polysome-associated ribosomes engated in protein synthesis.