Aleksandra Boguszewska

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.

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.

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.

Functional analysis of the uL11 protein impact on translational machinery.

ABSTRACT 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.

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 Ca2+-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.

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.