Overexpression in Escherichia coli, purification, and characterization of recombinant 60S ribosomal acidic proteins from Saccharomyces cerevisiae

The 60S ribosomal subunits from Saccharomyces cerevisiae contain a set of four acidic proteins named YP1alpha, YP1beta, YP2alpha, and YP2beta. The genes for each were PCR amplified from a yeast cDNA library, sequenced, and expressed in Escherichia coli cells using two expression systems. The first system, pLM1, was used for YP1beta, YP2alpha, and YP2beta. The second one, pT7-7, was used for YP1alpha. Expression in both cases was under the control of a strong inducible T7 promoter. The amount of induced recombinant proteins in the host cells was around 10 to 20% of the total soluble bacterial proteins. A new protocol for purification of all four recombinant proteins was established. The preliminary steps of purification were done by ammonium sulfate precipitation (YP1alpha, YP1beta) or NH4Cl/ethanol extraction (YP2alpha, YP2beta). The recombinant proteins were then purified to apparent homogeneity by only two steps of classical chromatographies, ion exchange (DEAE-cellulose) and gel filtration (Sephacryl S-200). Isoelectrofocusing analysis of YP2alpha and YP2beta showed the pIs of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pI of YP1alpha is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1beta was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1beta'. This was proved by isoelectrofocusing gel analysis.     

Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.     

The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1α/YP2β pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1α and YP2α proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1α/β defective strain, where a helping effect on the binding of the YP2α and YP2β proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.     

In vivo analysis of the acidic ribosomal proteins BmP1 and BmP2 of the silkworm Bombyx mori in the yeast Saccharomyces cerevisiae

In the silkworm Bombyx mori the ribosomal stalk P-protein family consists of two low MW acidic proteins, BmP1 and BmP2, and of one higher MW protein, BmP0, as shown by electrophoretical and immunoblotting western blot analysis of purified ribosomes. Treatment of ribosomes with alkaline phosphatase followed by electrofocusing shifted the isoelectric points to higher pH, implying phosphorylation of the proteins. The cDNAs encoding BmP1 and BmP2 proteins were constructed and expressed in the Saccharomyces cerevisiae mutant strains defective in either the endogenous P1 or P2 proteins. The recombinant silkworm proteins could complement the absence of the homologous yeast proteins and were incorporated to the ribosomes of the transformed strains, helping the binding of the remaining endogenous acidic proteins, present in the cytoplasm in different extent. Thus, BmP1 was able to replace YP1alpha, preferentially binding YP2beta to the ribosome, while BmP2 replaced both yeast P2 proteins and induced the binding of both YP1alpha and YP1beta.     

Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis.

The genes encoding the four acidic ribosomal phosphoproteins have been inactivated in Saccharomyces cerevisae by recombination with truncated genes carrying different genetic markers. By crossing single haploid disruptants, strains harboring two simultaneously inactivated acidic protein genes were constructed. None of the six possible double disruptions was lethal, but the simultaneous inactivation of either YP1 alpha and YP1 beta(L44') or YP2 alpha(L44) and YP2 beta(L45) caused an important decrease in the cell growth rate. Ribosomes isolated from these slow-growing strains did not contain acidic proteins, not even the two polypeptides whose genes were still intact, although these proteins were present in the cell extracts and they seem to be able to form high-molecular weight protein complexes. Transformation of a slow-growing double transformant with a plasmid containing one of the disrupted genes restored the presence of the acidic proteins in the ribosomes and normal growth rates. The particles of the slow-growing strains were active in an in vitro amino acid polymerizing system, although their activity could be stimulated by the exogenous addition of the missing proteins. These results indicate that in the absence of either YP1 alpha and YP1 beta(L44') or YP2 alpha (L44) and YP2 beta(L45), the remaining acidic proteins are unable to interact with the ribosome in a stable manner, but that a strong interaction of these ribosomal components with the particle is not an absolute requirement for in vivo and in vitro protein synthesis.     

Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans

In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit-the so-called stalk-structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal P-proteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and two-hybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B-P2A and P1A-P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins.     

Subcellular distribution of the acidic ribosomal P-proteins from Saccharomyces cerevisiae in various environmental conditions

The yeast ribosomal "stalk"--a lateral protuberance on the 60S subunit--consists of four acidic P-proteins, P1A, P1B, P2A and P2B, which play an important role during protein synthesis. Contrary to most ribosomal proteins, which are rapidly degraded in the cytoplasm, P-proteins are found as a cytoplasmic pool and are exchanged with the ribosome-bound proteins during translation. As yet, subcellular trafficking of P-proteins has not been extensively investigated. Therefore, we have characterized--using immunological approaches--the cellular distribution of P-proteins in several environmental conditions, characteristic of yeast cells, such as growth phases, and heat-, osmotic-, and oxygen-stress. Using the western blotting approach, we have shown P-proteins to be present in constant amounts on the ribosomes, despite their exchangeability with the cytoplasmic pool, and regardless of environmental conditions. On the other hand, P-protein level in the cytoplasm decreased sharply throughout the consecutive growth phases, but was not affected by several stress conditions. Applying the electron microscopic technique and immunogold labeling, we have found that P-proteins are located in two cell compartments. The first one is the cytoplasm and the second one--an unexpected place--the cell wall, where P-proteins are fully phosphorylated. Moreover, the existence of P-proteins on the cellular wall is not affected by various environmental conditions.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Elevated copy number of L-A virus in yeast mutant strains defective in ribosomal stalk

The eukaryotic ribosomal stalk, composed of the P-proteins, is a part of the GTPase-associated-center which is directly responsible for stimulation of translation-factor-dependent GTP hydrolysis. Here we report that yeast mutant strains lacking P1/P2-proteins show high propagation of the yeast L-A virus. Affinity-capture-MS analysis of a protein complex isolated from a yeast mutant strain lacking the P1A/P2B proteins using anti-P0 antibodies showed that the Gag protein, the major coat protein of the L-A capsid, is associated with the ribosomal stalk. Proteomic analysis revealed that the elongation factor eEF1A was also present in the isolated complex. Additionally, yeast strains lacking the P1/P2-proteins are hypersensitive to paromomycin and hygromycin B, underscoring the fact that structural perturbations in the stalk strongly influence the ribosome function, especially at the level of elongation.     

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.     

The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins.

The covalent structures of rat ribosomal proteins P0, P1, and P2 were deduced from the sequences of nucleotides in recombinant cDNAs. P0 contains 316 amino acids and has a molecular weight of 34,178; P1 has 114 residues and a molecular weight of 11,490: and P2 has 115 amino acids and a molecular weight of 11,684. The rat P-proteins have a near identical (16 of 17 residues) sequence of amino acids at their carboxyl termini and are related to analogous proteins in other eukaryotic species. A proposal is made for a uniform nomenclature for rat and yeast ribosomal proteins.     

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Background

The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.

Methods

We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.

Results

All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂ form represents the most favorable architecture for parasite P-proteins.

Conclusion

The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.

General significance

On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.     

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.     

Structural characterization of the ribosomal P1A-P2B protein dimer by small-angle X-ray scattering and NMR spectroscopy

The five ribosomal P-proteins, denoted P0-(P1-P2)2, constitute the stalk structure of the large subunit of eukaryotic ribosomes. In the yeast Saccharomyces cerevisiae, the group of P1 and P2 proteins is differentiated into subgroups that form two separate P1A-P2B and P1B-P2A heterodimers on the stalk. So far, structural studies on the P-proteins have not yielded any satisfactory information using either X-ray crystallography or NMR spectroscopy, and the structures of the ribosomal stalk and its individual constituents remain obscure. Here we outline a first, coarse-grained view of the P1A-P2B solution structure obtained by a combination of small-angle X-ray scattering and heteronuclear NMR spectroscopy. The complex has an elongated shape with a length of 10 nm and a cross section of approximately 2.5 nm. 15N NMR relaxation measurements establish that roughly 30% of the residues are present in highly flexible segments, which belong primarily to the linker region and the C-terminal part of the polypeptide chain. Secondary structure predictions and NMR chemical shift analysis, together with previous results from CD spectroscopy, indicate that the structured regions involve alpha-helices. NMR relaxation data further suggest that several helices are arranged in a nearly parallel or antiparallel topology. These results provide the first structural comparison between eukaryotic P1 and P2 proteins and the prokaryotic L12 counterpart, revealing considerable differences in their overall shapes, despite similar functional roles and similar oligomeric arrangements. These results present for the first time a view of the structure of the eukaryotic stalk constituents, which is the only domain of the eukaryotic ribosome that has escaped successful structural characterization.