Phosphorylation of the N-terminal domain of ribosomal P-stalk protein uL10 governs its association with the ribosome.

The uL10 protein is the main constituent of the ribosomal P-stalk, anchoring the whole stalk to the ribosome through interactions with rRNA. The P-stalk is the core of the GTPase-associated center (GAC), a critical element for ribosome biogenesis and ribosome translational activity. All P-stalk proteins (uL10, P1, and P2) undergo phosphorylation within their C termini. Here, we show that uL10 has multiple phosphorylation sites, mapped also within the N-terminal rRNA-binding domain. Our results reveal that the introduction of a negative charge within the N terminus of uL10 impairs its association with the ribosome. These findings demonstrate that uL10 N-terminal phosphorylation has regulatory potential governing the uL10 interaction with the ribosome and may control the activity of GAC.

Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation.

The P-stalk represents a vital element within the ribosomal GTPase-associated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2)2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo, especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Functional analysis of the uL11 protein impact on translational machinery.

The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

Molecular behavior of human Mrt4 protein, MRTO4, in stress conditions is regulated by its C-terminal region.

Protein Mrt4 is one of trans-acting factors involved in ribosome biogenesis, which in higher eukaryotic cells contains a C-terminal extension similar to the C-terminal part of ribosomal P proteins. We show that human Mrt4 (hMrt4/MRTO4) undergoes phosphorylation in vivo and that serines S229, S233, and S235, placed within its acidic C-termini, have been phosphorylated by CK2 kinase in vitro. Such modification does not alter the subcellular distribution of hMrt4 in standard conditions but affects its molecular behavior during ActD induced nucleolar stress. Thus, we propose a new regulatory element important for the stress response pathway connecting ribosome biogenesis with cellular metabolism.

Gene cloning, expression, and characterization of mutanase from Paenibacillus curdlanolyticus MP-1.

Mutanases hydrolyze d-glucosidic linkages of α-1,3-linked polysaccharides which are important components of dental plaque. Therefore, these enzymes can be useful in preventive oral hygiene. A gene encoding mutanase was cloned from soil-isolated Paenibacillus curdlanolyticus MP-1 and expressed in Escherichia coli, and the resulting recombinant enzyme was characterized. The nucleotide sequence of the mutanase gene consisted of 3786 nucleotides encoding a protein of 1261 amino acids with a theoretical molecular weight of 131.62kDa. The deduced amino acid sequence exhibited a high degree of similarity with mutanases of Paenibacillus sp. KSM-M126 and Paenibacillus humicus NA1123, with 84% and 80% identity, respectively. The recombinant enzyme was purified 17.5-fold to homogeneity with a recovery of 37%. The purified mutanase showed optimal activity at pH 6.0 and 45°C, and was completely stable at pH 4.0-9.5 and up to 45°C. The enzyme was specific for α-1,3-glucosidic linkages and effectively solubilized fungal α-1,3-glucans and streptococcal mutans, releasing nigerooligosaccharides. The mutanase did not hydrolyze a synthetic substrate readily hydrolyzed by exoglucanases and the enzyme activity was not suppressed in the presence of deoxynojirimycin, an inhibitor of exo-type enzymes. These results suggest an endohydrolytic mode of action.

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.

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.

Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins.

The ribosome has a distinct lateral protuberance called the stalk; in eukaryotes it is formed by the acidic ribosomal P-proteins which are organized as a pentameric entity described as P0-(P1-P2)(2). Bilateral interactions between P0 and P1/P2 proteins have been studied extensively, however, the region on P0 responsible for the binding of P1/P2 proteins has not been precisely defined. Here we report a study which takes the current knowledge of the P0 - P1/P2 protein interaction beyond the recently published information. Using truncated forms of P0 protein and several in vitro and in vivo approaches, we have defined the region between positions 199 and 258 as the P0 protein fragment responsible for the binding of P1/P2 proteins in the yeast Saccharomyces cerevisiae. We show two short amino acid regions of P0 protein located at positions 199-230 and 231-258, to be responsible for independent binding of two dimers, P1A-P2B and P1B-P2A respectively. In addition, two elements, the sequence spanning amino acids 199-230 and the P1A-P2B dimer were found to be essential for stalk formation, indicating that this process is dependent on a balance between the P1A-P2B dimer and the P0 protein.

Acquisition of a stable structure by yeast ribosomal P0 protein requires binding of P1A-P2B complex: in vitro formation of the stalk structure.

Saccharomyces cerevisiae ribosomal stalk consists of five proteins: P0 protein, with molecular mass of 34 kDa, and four small, 11 kDa, P1A, P1B, P2A and P2B acidic proteins, which form a pentameric complex P0-(P1A-P2B)/(P1B-P2A). This structure binds to a region of 26S rRNA termed GTPase-associated domain and plays a crucial role in protein synthesis. The consecutive steps leading to the formation of the stalk structure have not been fully elucidated and the function of individual P-proteins in the assembling of the stalk and protein synthesis still remains elusive. We applied an integrated approach in order to examine all the P-proteins with respect to stalk assembly. Several in vitro methods were utilized to mimic protein self-organization in the cell. Our efforts resulted in reconstitution of the whole recombinant stalk in solution as well as on the ribosomal particle. On the basis of our analysis, it can be inferred that the P1A-P2B protein complex may be regarded as the key element in stalk formation, having structural and functional importance, whereas P1B-P2A protein complex is implicated in regulation of stalk function. The mechanism of quaternary structure formation could be described as a sequential co-folding/association reaction of an oligomeric system with P0-(P1A-P2B) protein complex as an essential element in the acquisition of a stable quaternary structure of the ribosomal stalk. On the other hand, the P1B-P2A complex is not involved in the cooperative stalk formation and our results indicate an increased rate of protein synthesis due to the latter protein pair.

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.

Structural characterization of yeast acidic ribosomal P proteins forming the P1A-P2B heterocomplex.

Acidic ribosomal P proteins form a distinct lateral protuberance on the 60S ribosomal subunit. In yeast, this structure is composed of two heterocomplexes (P1A-P2B and P1B-P2A) attached to the ribosome with the aid of the P0 protein. In solution, the isolated P proteins P1A and P2B have a flexible structure with some characteristics of a molten globule [Zurdo, J., et al. (2000) Biochemistry 39, 8935-8943]. In this report, the structure of P1A-P2B heterocomplex from Saccharomyces cerevisiae is investigated by means of size-exclusion chromatography, chemical cross-linking, circular dichroism, light scattering, and fluorescence spectroscopy. The circular dichroism experiment shows that the complex could be ranked in the tertiary class of all-alpha proteins, with an average alpha-helical content of approximately 65%. Heat and urea denaturation experiments reveal that the P1A-P2B complex, unlike the isolated proteins, has a full cooperative transition which can be fitted into a two-state folding-unfolding model. The behavior of the complex in the presence of 2,2,2-trifluoroethanol also resembles a two-state folding-unfolding transition, further supporting the idea that the heterocomplex contains well-packed side chains. In conclusion, the P1A-P2B heterocomplex, unlike the isolated proteins, has a well-defined hydrophobic core. Consequently, the complex can put up its structure without additional ribosomal components, so the heterodimeric complex reflects the intrinsic properties of the two analyzed proteins, indicating thus that this is the only possible configuration of the P1A and P2B proteins on the ribosomal stalk structure.

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.