PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress.

The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)2, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.

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)2form 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.

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein.

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10ß. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10ß protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10ß is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10ß undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10ß, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2 of Plasmodium falciparum in a Mouse Model.

Malaria remains one the most infectious and destructive protozoan diseases worldwide. Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins from P. falciparum and determined their immunogenicity in an in vivo assay on a mouse model. The pentameric complex P0-(P1-P2)2 was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-ß. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2 were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. An in vitro exposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2 protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.

The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity.

The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.

Functional Analysis of the Ribosomal uL6 Protein of Saccharomyces cerevisiae.

The genome-wide duplication event observed in eukaryotes represents an interesting biological phenomenon, extending the biological capacity of the genome at the expense of the same genetic material. For example, most ribosomal proteins in Saccharomyces cerevisiae are encoded by a pair of paralogous genes. It is thought that gene duplication may contribute to heterogeneity of the translational machinery; however, the exact biological function of this event has not been clarified. In this study, we have investigated the functional impact of one of the duplicated ribosomal proteins, uL6, on the translational apparatus together with its consequences for aging of yeast cells. Our data show that uL6 is not required for cell survival, although lack of this protein decreases the rate of growth and inhibits budding. The uL6 protein is critical for the efficient assembly of the ribosome 60S subunit, and the two uL6 isoforms most likely serve the same function, playing an important role in the adaptation of translational machinery performance to the metabolic needs of the cell. The deletion of a single uL6 gene significantly extends the lifespan but only in cells with a high metabolic rate. We conclude that the maintenance of two copies of the uL6 gene enables the cell to cope with the high demands for effective ribosome synthesis.

Mechanism of completion of peptidyltransferase centre assembly in eukaryotes.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

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.

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.

Subcellular localization of ribosomal P0-like protein MRT4 is determined by its N-terminal domain.

The Mrt4 protein, showing extensive sequence similarity to the ribosomal P0 protein, is classified as a ribosomal P0-like protein and acts as a trans-acting factor which modulates the assembly of the pre-60S particle. In this report we investigated the biological nature of the human Mrt4 protein. First, we constructed a series of hybrid hMrt4-P0 proteins by replacing various domains of the P0 protein with corresponding protein fragments from hMrt4. We found that hMrt4 binds to the same site on the large ribosomal subunit as does P0, but despite the sequence homology it is not able to functionally complement the lack of P0. Using fluorescence microscopy and biochemical approaches we also show that hMrt4 occupies predominantly the nucleolar compartment, in contrast to P0 and P1/P2, which are located in the cytoplasm. The nucleolar accumulation of hMrt4 does not depend on a specific nucleolus localization signal, but rather occurs via interaction with established nucleolar components such as rRNA; however, nuclear import of hMrt4 is dependent on a short sequence in the N-terminal part of the protein. Functional analysis with specific inhibitors, actinomycin D and leptomycin B, showed that hMrt4 is a trans-acting factor involved in ribosome maturation, with nucleus-cytoplasm shuttling capacity.