Mouse myeloma cells (MPC 11) in culture, labelled with I3'P]i for several hours, were subjected to hypertonic initiation block by raising the salt concentration of the culture medium from 110 to 210 mM NaCl.The relative amounts of messenger-free and bound ribosomes of the cells were estimated from ribosome profiles following sucrose density centrifugation. The initiation block caused the proportion of messenger-bound ribosomes to decrease substantially. The extent of the decrease was larger under suboptimal (50 %) than under satisfactory (25 %) nutritional conditions. Analysis of the ribosomal proteins by two-dimensional gel electrophoresis, autoradiography and radiospectrometry showed, that in the small subunit only protein S3 had incorporated 32P and that S3 was the most strongly phosphorylated ribosomal protein in control cells. Upon salt treatment of the cells the radioactivity of S3 from both messenger-free and bound ribosomes decreased to low levels. The lowest levels (15% of the controls) were reached when the nutritional conditions were suboptimal. The results show that there is a positive correlation between the number of ribosomes engaged in protein synthesis and the degree of phosphorylation of S3. However, phosphorylation of S3 does not seem to be a prerequisite for ribosomes to be attached to mRNA. Under all experimental conditions S3 from messenger-bound ribosomes was about twice as strongly labelled as S3 from messenger-free ribosomes.The large-subunit phosphoproteins were identified as L28, the protein thought to be homologous to the Escherichia coli proteins L7/L12, and as L5, which was only weakly labelled. In contrast to S3 the radioactivity of L28 increased slightly upon the tonicity shift.Of the approximately 70 different species of ribosomal proteins from eukaryotic cells some 4 or 5 are phosphorylated in vivo [1,2] of which at least two, namely the small-subunit protein S3 (denoted S6 in the nomenclature of Sherton and Wool [3]) and the acidic, large-subunit protein(s) (likely to be homologous to proteins L7/L12 of Escherichia coli [4]) can become phosphorylated at more than one site ([5,6] and [7,8]
respectively).As to the biological significance of ribosomal protein phosphorylation, neither experiments in vitro [9 -11 ] nor the numerous studies in vivo [5,6, have yielded conclusive results. In particular, it has remained controversial to which, if any, parameter of protein metabolism phosphorylation of ribosomal proteins may be related [5,13,15,17,18].Abbreviations. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Tris/K/Mg, buffer containing 50 mM Tris-HCI, pH 7.6, 25 mM KCI, 5 mM magnesium acetate, 1 mM dithiothreitol.Of the three principal stages of peptide chain synthesis, initiation has generally been regarded the most likely to be under regulatory control. Experimentally, the frequency of peptide chain initiation can be drastically reduced in cultured cells by increasing the tonicity of their growth medium, socalled hypertonic initiation block 119 -221. Because of the...
