Nikodem Grankowski

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.

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.

Structural Relationships Among the Ribosomal Stalk Proteins from the Three Domains of Life

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.

Non-AUG translation initiation of mRNA encoding acidic ribosomal P2A protein in Candida albicans

The eukaryotic 60S ribosomal subunit has a set of very acidic proteins (P‐proteins), which form a distinct lateral protuberance called the stalk structure. This protein complex is directly involved in the elongation step of polypeptide synthesis. In our study on acidic ribosomal P‐proteins from the human opportunistic pathogen Candida albicans, we isolated and characterized one of the genes, called CARP2A, and its product, the P2A protein. The CARP2A gene is intron‐less, present in a single copy per haploid genome, and transcriptionally active. The open reading frame of the studied gene contains information for a sequence of 108 amino acids. Based on this, the molecular mass and isoelectric point of the P2A protein were theoretically calculated to be 10.85 kDa and 3.7, respectively. The characteristic feature of the CARP2A gene transcript is the presence of a GUG start codon, which is rare in eukaryotic organisms and not previously reported in yeast. To our knowledge this is the first report showing the presence of a naturally occurring non‐AUG start codon on mRNA in yeast species. The nucleotide sequence of CARP2A has been deposited in the GenBank data library under Accession No. AF317661. Copyright

Biophysical Properties of the Eukaryotic Ribosomal Stalk

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

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.

Subcellular localization of casein kinase I.

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.

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.

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein substrate not only depends on the structure of the polypeptide chain around the target amino acid but also on its native structure within the 80S ribosome.

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.