Phosphorylation of a 40S ribosomal subunit protein in Tetrahymena

Abstract

In Tetrahymena the small ribosomal subunit protein S7, which appears to be the equivalent of S6 of higher eukaryotes, undergoes reversible phosphorylation under a set of defined conditions. In an attempt to understand the physiological role of such reversible phosphorylation, we examined the status of ribosomal protein S7 in growing cells and growth-arrested cells, starving either non-specifically for nutrients or specifically for a single essential amino acid. These experiments allowed us to dissociate S7 phosphorylation from changes in the translational activity and the stability of ribosomes. The results revealed complete lack of correlation between phosphorylation of S7 and both the growth status of the cells and the in vivo stability of ribosomes. Taken together with the observation that phosphorylation of S7 occurs only when the cells are starved in buffers containing sodium chloride or high concentrations of Tris, non-essential ions for normal growth, our data suggest that this protein modification is required to maintain the functional integrity of the ribosomes in an altered electrostatic environment, induced by changes in the extracellular ionic conditions. Reversible and multiple phosphorylation (2 -5 phosphate residues per molecule) of the small-ribosomal-subunit protein S6, ubiquitous in eukaryotic cells, is thought to play a role in the regulation of the initiation of protein synthesis [l -31; because this ribosomal protein modification appears to confer a selective advantage for entry of ribosomes into polysomes [l]. Moreover, there is a temporal and quantitative correlation between the increase in the rate of protein synthesis and the degree of S6 phosphorylation in growth-stimulated cells [2,4]. Nevertheless, the biological significance of S6 phosphorylation, particularly its role in the regulation or even determination of the rate of protein synthesis, still remains controversial; thus, Kruse et al. [5] have recently demonstrated that in yeast, phosphorylation of S10, the yeast equivalent of S6, is not indispensable for growth; moreover, in the ciliate protozoa, Tetrahymena, such phosphorylation is absent in the actively growing cells but is induced when these cells are transferred to nutrient-free dilute salt solutions [6 -81, conditions in which cellular proliferation is arrested, protein synthesis is drastically curtailed and ribosome degradation is turned on [9, lo]. These events, including the ribosomal protein phosphorylation, are rapidly reversed upon refeeding the starved cells [S, 91. Since phosphorylation of a smallribosomal-subunit protein and decay of ribosomes not only occur together upon transfer of growing Tetrahymena to starvation buffers but also display similar kinetics, Kristiansen and Kriiger [ll] have suggested that this protein modification might be involved in the regulation of turnover of ribosomes. Such a role has also been suggested for the multiple phosphorylation of S6 in the rat liver [12].With respect to its molecular mass, electrophoretic mobility and capacity...

Monovalent cation‐dependent reversible phosphorylation of a 40 S ribosomal subunit protein in growth‐arrested Tetrahymena: correlation with changes in intracellular pH

“…It is therefore possible that in hormone or mitogen stimulated mammalian cells, increase in pHi and phosphorylation of S6 may be independent manifestations of the effect of these stimuli on cell metabolism. The present study shows that, in T. thermoph~la, phosphorylation and dephosphorylation of ribosomal protein S8 can be induced in starved cells which, as we have previously shown [8,24], do not divide and display a slow net decline in their total RNA content, and a rapid reduction in their rate of protein synthesis. Under these conditions, which differ greatly from those pertaining in growth-stimulated cells, phosphorylation of protein S8 and an increase in pHi are induced together by addition of Na+ to the cell suspension medium, and the two effects are reversed together by subsequent addition of K+.…”