Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.     

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.     

Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth.

The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes. Its potential role in the regulation of protein synthesis has been the object of much study. We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis. The S10 of such cells is not phosphorylated. Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock. We conclude that in S. cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins. This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.