Role of ribosomal subunits in protein synthesis in mammalian cells.

Exchange of ribosomal subunits (RSU) in the course of protein synthesis has been demonstrated in bacteria." 2 RSU have been shown to be stable during bacterial growth and to be continuously recycled through ribosomes.2 These results suggested that ribosomes dissociate into subunits between successive rounds of protein synthesis. The 30S E. coli RSU binds the initiator tRNA (formylmethionine-tRNA) in the presence of f2-RNA3 or of the synthetic polynucleotide poly r-AUG or poly r-UG.4 Subsequent addition of a 50S subunit results in the formation of a complex which binds noninitiator tRNA and is capable of carrying out peptide synthesis then supplemented with the soluble components of the protein-synthesizing system.3' 4 The 70S ribosomes and RSU derived from 70S ribosomes are much less active than native RSU in supporting polypeptide synthesis directed by f2-RNA.6 The 70S ribosomes of E. coli and the derived subunits appear to be deficient in an initiation factor,6'7 which is associated with the 30S RSU.6 The simplest explanation of RSU exchange, of the specific binding of messenger RNA to the 30S subunit, and of the association of the initiation factor with the 30S native subunit is that dissociation of ribosomes into subunits and association of native subunits with messenger RNA are necessary steps in protein synthesis. Subunit exchange has not yet been shown to occur in eucaryotic cells. The addition of native RSU to a cell-free system from reticulocytes enhances the initiation of new globin chains.8 It may thus be inferred that RSU are involved in chain initiation in these cells. The present study was undertaken with the aim of demonstrating the role of RSU in protein synthesis in intact eucaryotic cells. Inhibition of protein synthesis by NaF in reticulocytes is known to cause polyribosome disaggregation and accumulation of 80S ribosomes.9 NaF appears to inhibit specifically initiation of new chains, whereas it does not inhibit chain completion'0 11 or chain reinitiation on ribosomes already attached to messenger RNA after removal of peptidyl-tRNA by puromycin.'2 The present experiments show that NaF causes a decrease in the level of RSU preceding the inhibition of protein synthesis in reticulocytes and in HeLa cells. After removal of NaF, reappearance of RSU precedes the recovery from inhibition of protein synthesis. These experiments suggest that the presence of RSU is necessary for initiation of chain synthesis and also that NaF may interfere with the dissociation of 80S ribosomes into 60S and 40S subunits. Materials and Methods.-Preparation and incubation of cells: Reticulocytes were obtained from phenylhydrazine-injected rabbits according to the procedures of Borsook et al.'3 Washing of reticulocytes and conditions for incubation have been previously described.'4 [C14] amino acid mixture (New England Nuclear) was used in incorporation