Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

Abstract

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...

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

“…In addition to P-proteins P0, P1, and P2, plants possess an additional P1/P2 type protein termed P3 (56). The P-proteins were initially identified as phosphoproteins in the yeast ribosome (57), and their phosphorylation states likely play a direct role in protein synthesis and translational responses to external stimuli (26,58). Phosphorylation sites in the yeast P-proteins occur in the C-terminal region of the proteins (58), a feature that has now been confirmed for all members of the Arabidopsis family of ribosomal P-proteins.…”

“…Covalent Modifications of Arabidopsis Cytosolic Ribosomal Proteins-Covalent protein modifications such as acetylation, methylation, and phosphorylation have emerged as potentially being important factors contributing to ribosomal heterogeneity in both eukaryotes (4,19,26,27) and prokaryotes (28,29). In the ribosomes of higher plants, studying the role of covalent r-protein modification in ribosomal heterogeneity is complicated by the frequent expression of relatively high (compared with other classes of organism) numbers of different, yet often highly conserved isoforms of each ribosomal subunit.…”

“…The C terminus of RPS6 is sequentially phosphorylated on five serine residues in response to a variety of stimuli and developmental cues (53,54). This C-terminal region is highly conserved across eukaryotic lineages, and in plants its mul-tiphosphorylation sites have been well characterized in maize and Arabidopsis (26,27). Initial characterization of this region in mammals has demonstrated an ambiguity in phosphorylation site determination (54).…”

The ribosome filter hypothesis

“…In maize and yeast, a set of very acidic ribosomal proteins (P-proteins) that are associated with 60S ribosomal subunits are heterogeneous (62,63). In yeast, ribosomes from stationary phase are deficient in P-proteins when compared with those from exponentially growing cells (63).…”

Plant L10 Ribosomal Proteins Have Different Roles during Development and Translation under Ultraviolet-B Stress

“…Nearly half are represented by two or more protein spots on 2D gels, indicating that proteins are posttranslationally modified and/or present as different isoforms (Chang et al, 2005;Giavalisco et al, 2005;Carroll et al, 2008). It is hypothesized that this ribosomal heterogeneity fosters selective translation of specific mRNA under particular cell conditions (Barakat et al, 2001;Szick-Miranda and Bailey-Serres, 2001;Giavalisco et al, 2005;Carroll et al, 2008).…”