Hendrik A. Raué

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.

Identification and functional analysis of the nuclear localization signals of ribosomal protein L25 from Saccharomyces cerevisiae

The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase. Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus. Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself. Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein. This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25. The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits. Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.

Yeast Rrp9p is an evolutionarily conserved U3 snoRNP protein essential for early pre-rRNA processing cleavages and requires box C for its association

Pre-rRNA processing in eukaryotic cells requires participation of several snoRNPs. These include the highly conserved and abundant U3 snoRNP, which is essential for synthesis of 18S rRNA. Here we report the characterization of Rrp9p, a novel yeast U3 protein, identified via its homology to the human U3-55k protein. Epitope-tagged Rrp9p specifically precipitates U3 snoRNA, but Rrp9p is not required for the stable accumulation of this snoRNA. Genetic depletion of Rrp9p inhibits the early cleavages of the primary pre-rRNA transcript at A0, A1, and A2 and, consequently, production of 18S, but not 25S and 5.8S, rRNA. The hU3-55k protein can partially complement a yeast rrp9 null mutant, indicating that the function of this protein has been conserved. Immunoprecipitation of extracts from cells that coexpress epitope-tagged Rrp9p and various mutant forms of U3 snoRNA limits the region required for association of Rrp9p to the U3-specific box B/C motif. Box C is essential, whereas box B plays a supportive role.

Effect of a pmr1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba α-galactosidase by Saccharomyces cerevisiae

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.

The roles of Rrp5p in the synthesis of yeast 18S and 5.8S rRNA can be functionally and physically separated

The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

The cellular level of yeast ribosomal protein L25 is controlled principally by rapid degradation of excess protein

SummaryWhen the gene dosage for the primary rRNA-binding ribosomal protein L25 in yeast cells was raised about 50-fold, the level of mature L25 transcripts was found to increase almost proportionally. The plasmid-derived L25 transcripts were structurally indistinguishable from their genomic counterparts, freely entered polysomes in vivo and were fully translatable in a heterologous in vitro system. Nevertheless, pulse-labelling for periods varying from 3–20 min did not reveal a significant elevation of the intracellular level of L25 protein. When pulse-times were decreased to 10–45 s, however, we did detect a substantial over production of L25. We conclude that, despite the strong RNA-binding capacity of the protein, accumulation of L25 is not controlled by an autogenous (pre-)mRNA-targeted mechanism similar to that operating in bacteria, but rather by extremely rapid degradation of excess protein produced.

Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E. coli 23S rRNA.

The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments. The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465‐1632 and 1811‐1861. The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site. This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA. In fact heterologous complexes between L25 and E. coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure. Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E. coli ribosomal protein EL23. Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.

Nuclear and nucleolar localization of Saccharomyces cerevisiae ribosomal proteins S22 and S25

Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS. Comparison with previously published data on yeast ribosomal protein NLSs and computer analysis indicates the existence of a novel type of ribosomal protein‐specific NLS that differs from the classical Chelsky and bipartite NLSs. The existence of such a ribosomal protein‐specific NLS is in accordance with the recent identification of ribosomal protein‐specific importins.

In vivo and in vitro analysis of structure-function relationships in ribosomal protein L25 from Saccharomyces cerevisiae

We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.

rRNA binding domain of yeast ribosomal protein L25: Identification of its borders and a key leucine residue☆

We have delineated the region of yeast ribosomal protein L25 responsible for its specific binding to 26 S rRNA by a novel approach using in vitro synthesized, [35S]methionine-labeled fragments as well as point mutants of the L25 protein. The rRNA binding capacity of these mutant polypeptides was tested by incubation with an in vitro transcribed, biotinylated fragment of yeast 26 S rRNA that contains the complete L25 binding site. Protein-rRNA interaction was assayed by binding of the rRNA-r-protein complex to streptavidin-agarose followed either by analysis of the bound polypeptide by SDS/polyacrylamide gel electrophoresis or by precipitation with trichloroacetic acid. Our results show that the structural elements necessary and sufficient for specific interaction of L25 with 26 S rRNA are contained in the region bordered by amino acids 62 and 126. The remaining parts of the protein, in particular the C-terminal 16 residues, while not essential for binding, do enhance its affinity for 26 S rRNA. To test whether, as suggested by the results of the deletion experiments, the evolutionarily conserved sequence motif K120KAYVRL126 is involved in rRNA binding, we replaced the leucine residue at position 126 by either isoleucine or lysine. The first substitution did not affect binding. The second, however, completely abolished the specific rRNA binding capacity of the protein. Thus, Leu126, and possibly the whole conserved sequence motif, plays a key role in binding of L25 to 26 S rRNA.