Rabbit reticulocyte suspensions extensively incorporate [32P]phosphate into covalent linkage with ribosome structural proteins. Only a minor fraction of the incorporation is into ribonucleic acids or their terminal nucleotides (less than 1-2 %) or is into phospholipids (less than 10 %). At least 75 % of the ribosome-bound phosphate is released from the ribosomes by digestion with Escherichia coli alkaline phosphomonoesterase. The phosphate occurs in the ribosomes as o-phosphoserine and o-phosphothreonine residues of polypeptides. A similar incorporation into reticulocyte ribosomal phosphoproteins occurs in vivo after injection of rabbits with [32P]phosphate. All tested chick embryo tissues (heart, liver, muscle) also contain phosphoproteins in their ribosomes. The radioactive ribosomal phosphoproteins have been analyzed by electrophoresis in polyacrylamide gels containing 0.6% sodium dodecyl sulfate. The ribosomes fully dissociate in the gels and all of the macromolecular components migrate toward the anode. The mobilities of protein-dodecyl sulfate complexes are inversely proportional to the logarithms of the polypeptide chain molecular weights. All of the major radioactive components are converted into rapidly migrating low molecular weigh-material by proteolytic digestion with pronase. The following conclusions can be made about the phosphorylated components of ribosomes. (1) Approximately 40% of the radioactivity associated with ribosomes is present in about 10 different loosely associated phosphoproteins. These components are probably contaminants since they can be removed by washing the ribosomes in high ionic strength solution. Furthermore, these components are present on polyribosomes, single ribosomes, subribosomal particles, and also in the supernatant fraction. (2) The "P band" of the gels contains a phosphorylated compound which is specifically and firmly associated only with polysomal ribosomes and which has a rapid electrophoretic mobility. The P component is released quantitatively from polysomal ribosomes as they are converted into single ribosomes during reticulocyte incubation with 0.03 M NaF. Dissociation of ribosomes into subunits with EDTA also causes release of P. The molecular nature of P is uncertain. (3) The "Si band" is a protein (mol wt ∼21,000 daltons) which is present on the larger ribosomal subunits. Although present also on polysomes and on subribosomal particles, the Si protein is only phosphorylated on single ribosomes. Phosphorylation of Si protein is not obligatorily coupled to the formation of single ribosomes which occurs when reticulocytes are incubated with 0.03 M NaF. The kinetics of Si phosphorylation suggest that the phosphate is turning over only slowly and that single ribosomes containing phosphorylated Si protein are excluded from participating in the ribosomal subunit-polyribosome cycle of protein synthesis. They are a class of inactive particles which may be a storage form of ribosomes. (4) The "Su band" is a large phosphoprotein (mol wt ∼70,000 daltons) which is specifically associated only with the small 44S native subribosomal particles. It is absent from the supernatant suggesting that it is not a contaminant of ribosomes. However, the unphosphorylated Su protein is also absent from polyribosomes and single ribosomes. The Su protein can be eluted from the subribosomal particles by washing them in high ionic strength solutions. (5) The "F band" is a phosphoprotein (mol wt ∼ 33,000 daltons) which is firmly associated with all ribosome fractions and which is absent from the supernatant fraction. The F protein is on the small ribosomal subunits. Phosphorylation of F is specifically stimulated when the cells are incubated with 0.03 M NaF, even when polyribosome disaggregation is blocked with cycloheximide. This effect of NaF is unrelated to the well-known enhancement by fluoride of the intracellular concentration of cyclic 3′,5′-adenosine monophosphate. It is concluded that eukaryote ribosomes are heterogeneous and are subject to modification by protein phosphorylation. This modification appears to regulate their ability to participate in protein synthesis.
|Original language||English (US)|
|Number of pages||16|
|Publication status||Published - 1970|
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