Rat RL23a ribosomal protein efficiently competes with its Saccharomyces cerevisiae L25 homologue for assembly into 60S subunits.

The large subunit protein RL23a from rat liver ribosomes shows 62% sequence identity with the primary rRNA-binding ribosomal protein L25 from Saccharomyces cerevisiae. In vitro binding studies indicated that both r-proteins are able to recognise the L25 binding site on yeast 25 S rRNA and its structural homologue on mammalian 28 S rRNA with equal efficiency. To determine whether the two r-proteins are also functionally equivalent in vivo, a single plasmid-borne copy of either the wild-type L25 gene or the RL23a cDNA, driven by the L25 promoter, was introduced into a yeast strain in which the chromosomal L25 gene is under control of the glucose-repressible GALI-10 promoter. No difference in growth rate could be detected between the two types of transformants when cultured on glucose-based medium. In cells that co-express epitope-tagged versions of L25 and RL23a from single-copy genes, approximately 35% of the 60 S subunits contained the heterologous protein as determined by Western analysis. This value could be increased to 55% by overexpressing RL23a using a multi-copy plasmid. These data demonstrate that rat RL23a can act as a highly efficient substitute for its yeast counterpart in the assembly of functional yeast ribosomes even in the presence of the endogenous L25 protein

Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast.

When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium. The remainder accumulates intracellularly in a misfolded conformation. We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells. Indeed, a tenfold increase in the lever of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin. By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced. Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression

Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function.

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991). Proc. Natl. Acad. Sci. USA 88, 3962–3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre‐rRNA operon under control of the inducible, Pol II‐transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose‐based medium making the cells completely dependent on plasmid‐encoded rRNA. We show that the Pol II‐transcribed pre‐rRNA is processed and assembled similarly to authentic Pol I‐synthesised pre‐rRNA, making this ‘in vivo Pol II system’ suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551–559], is in fact a lethal mutation. Copyright © 1995 John Wiley & Sons Ltd

Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit

The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the bilogical function of the 40S subunit