Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one-and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.The ribosome is a two-subunit ribonucleoprotein complex that catalyzes the peptidyl transferase reaction of polypeptide synthesis, an absolute requirement for cellular growth and differentiation. The structure and function of both prokaryotic and eukaryotic ribosomes have been investigated, with the eukaryotic emphasis on ribosomes of Baker's yeast (Saccharomyces cerevisiae) and rat (Rattus rattus and Rattus norvegicus). The cytosolic ribosomes of eukaryotes are composed of a large number of ribosomal proteins (r-proteins) and four distinct rRNAs, the 18S rRNA of the 40S subunit, and the 5S, 5.8S, and 23S-like (25-28S) rRNAs of the 60S subunit (Bielka, 1982). Early evaluation of the buoyant density and sedimentation coefficients of eukaryotic ribosomes predicted that the rat 80S ribosome has a higher mass (4.2-4.6 MD) than that of pea (Pisum sativum; 3.9 MD) due to distinctions in the 60S subunit (Cammarano et al., 1972). Further evidence that the plant ribosome is smaller than that of mammals was provided by the three-dimensional reconstruction of wheat (Triticum aestivum) and rabbit (Oryctolagus cuniculus) ribosomes by use of cryoelectron microscopy (Verschoor et al., 1996). Despite an overall similarity in architecture, the 60S subunit of wheat appeared approximately 20% smaller than that...

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.The eukaryotic ribosome is a complex structure composed of four rRNAs and about 80 ribosomal proteins (r-proteins). It represents an essential piece of the cell machinery, responsible for protein synthesis, and as such plays a major role in controlling cell growth, division, and development. For example, previous studies have shown that genetic defects in ribosomal components, such as reduction of the levels of individual r-proteins, can cause deleterious effects on the development and physiology of an organism. In Drosophila melanogaster, mutations in r-proteins genes cause the haplo-insufficient Minute phenotype with reduced growth and cell division rates, characterized by a reduced body size and short, thin bristles (Lambertsson, 1998). In contrast, a conditional deletion in the gene encoding r-protein S6 in adult mice (Mus musculus) affects cell cycle progression but not cell growth (Volarevic et al., 2000). In humans, a quantitative reduction in synthesis of the X-linked form of r-protein S4 is observed in individuals with Turner syndrome (monosomic for X) and may contribute to this complex phenotype, which includes short stature and infertility (Zinn and Ross, 1998). In plants, mutations in r-protein genes affect embryo viability or plant development (Van Lijsebettens et al., 1994;Tsugeki et al., 1996;Revenkova et al., 1999;Ito et al., 2000). In addition, a positive correlation was reported between the level of r-protein gene transcript accumulation and cell division i...

Maize (Zea mays L.) possesses four distinct ϳ12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.A complex of acidic ribosomal proteins (r-proteins) 1 forms a universally conserved lateral stalk on the large ribosomal subunit that facilitates the translocation phase of protein synthesis (1). In eukaryotes the structure is formed by a complex of acidic phosphoproteins. P0 (ϳ35 kDa), homologous to prokaryotic L10, interacts with 28 S rRNA to form the base of the stalk, and P1 and P2 (ϳ12 kDa), homologous to prokayotic L7/L12, are tethered as dimers to the stalk (2-5). P1 and P2 are structurally similar; each protein has three domains that include an ␣-helical N-terminal region and a central, flexible acidic hinge region followed by a highly conserved C terminus (E/KSD/ EDMGFG/SLD). The C-terminal region of P0 is structurally similar to 12-kDa P-proteins because it possesses the three domains of P1 and P2 (for review, see Ref. 6).The 12-kDa P-proteins are the only r-proteins found in multiple copies within the ribosome. They do not assemble onto preribosomes in the nucleolus but cycle between ribosomes and a cytosolic pool in numerous species including, Artemia salina, Saccharomyces cerevisiae (yeast), humans, and rats (7-11). demonstrated quantitatively that exponentially growing yeast cells contain more 12-kDa P-proteins/ribosome than cells in the stationary phase of growth. This suggests that the level of P-proteins in yeast ribosomes is affected by the metabolic state of the cell and possibly reflects the translational activity of the ribosome. The presence of these proteins in ribosomes has been shown to stimulate the eEF2-dependent GTPase activity of ribosomes (13-17), poly(U)-directed phenylalanine synthesis (14, 18), and eEF1A binding (19). Hence, modulation of the 12-kDa P-protein component of ribosomes may impar...

-Numerous changes in gene expression occur in response to flooding (oxygen deprivation, i.e., anoxia and hypoxia) in seedling roots of maize ( Zea mays L.) and other plants. Increased de novo transcription of anaerobic polypeptide (ANP) genes is responsible, in part for increased production of glycolytic and fermentative enzymes, such as alcohol dehydrogenase-1. There is increasing evidence that regulation of mRNA translation plays an important role in the expression of ANP genes during oxygen deprivation. By quantitative analysis of ribosomal complexes, we demonstrated a dramatic decrease in polysomes and an increase in monosomes in maize seedling roots deprived of oxygen, indicative of regulation of translational initiation. We report that oxygen deprivation causes dynamic changes in the phosphorylation status of the eukaryotic initiation factors (eIF) eIF4E, eIF4A, and eIF4B. By affinity purification of initiation complexes with 7m GTP and poly(A) resins, we demonstrate that a reduction in pH, which occurs in the cytosol in response to this stress, affects the assembly of mRNA 5'-cap and 3'-tail-binding complexes. We also describe oxygen deprivation-induced changes in phosphorylation of ribosomal protein S6, ribosomal 12-kD P-proteins and eukaryotic elongation factor-2 (eEF2). A model is presented that considers the implication of modifications in translational machinery in the interactions between the 5'-cap and 3'-tail of the mRNA that facilitate initiation and eEF2 GTPase activity which promotes elongation.