Regulated heterogeneity in 12-kDa P-protein phosphorylation and composition of ribosomes in maize (Zea mays L.).

Maize (Zea mays L.) possesses four distinct approximately 12-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.


INTRODUCTION
A complex of acidic ribosomal proteins (r-proteins) 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 (~35 kDa), homologous to prokaryotic L10, interacts with 28S rRNA to form the base of the stalk, and P1 and P2 (~12 kDa), homologous to prokayotic L7/L12, are tethered as dimers to the stalk (2,3,4,5). P1 and P2 are structurally similar; each protein has three domains that include an alphahelical N-terminal region, 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 since it possesses the three domains of P1 and P2 (reviewed in 6).
The 12-kDa P-proteins are the only r-proteins found in multiple copies within the ribosome.
They do not assemble onto pre-ribosomes in the nucleolus but cycle between ribosomes and a cytosolic pool in numerous species including, Artemia salina, Saccharomyces cerevisiae (yeast), humans and rats (7,8,9,10,11). Saenz-Robles et al. (12), demonstrated quantitatively, that exponentially growing yeast cells contain more 12-kDa P-proteins per 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 eEF2dependent GTPase activity of ribosomes (13,14,15,16,17), polyU-directed phenylalanine synthesis (14,18), and eEF1A binding (19). Hence, modulation of the 12-kDa P-protein component of ribosomes may impart eukaryotes with a means of ribosome regulated translational control.
Higher eukaryotes possess one type of P1 and P2, whereas lower eukaryotes possess multiple forms of P1 and P2 (20). S. cerevisiae ribosomes, for example, possess two forms of P1 (P1α and P1β) and P2 (P2α and P2β) (21). Mutant yeast strains in which one to four of the 12-kDa Pprotein genes were disrupted remained viable but had decreased rates of cell growth (22,23,20).
Ribosomes isolated from strains in which two or three genes were disrupted showed reduced eEF2-dependent GTPase activity and levels of protein synthesis in vitro (23). The strain in which all four genes were disrupted was more severely impaired, unable to produce spores, and cold sensitive. Remarkably, the profile of proteins synthesized from the same poly(A) mRNA sample with ribosomes from this strain and a wildtype strain were distinct, suggesting the presence of P1 and P2 influences the efficiency of translation of individual mRNAs.
P1 and P2 and their phosphorylated forms appear to be functionally distinct. In yeast, the presence of P1 was required for the assembly of P2 into the ribosome (23,24), and the ability to dephosphorylate one or more of these proteins was necessary for an adaptive response to osmotic stress (25). Studies with recombinant P1 and P2 from rat suggested that phosphorylation of P2 more effectively stimulates eEF2 activity in vitro than phosphorylation of P1 (17). Further analyses indicated that P1 has a higher binding affinity for eEF2, but that phosphorylation of both proteins stabilizes the interaction of eEF2 with the ribosome (16). These data suggest the presence and phosphorylation of the 12-kDa P-proteins is involved in ribosome mediated translational regulation.
We reported that maize (Zea mays L.) possesses one form of P1, two forms of P2 (P2a and P2b) and a third, plant-specific P1/P2-type protein designated P3 (26,27). Here we examined whether maize ribosomes vary with respect to the composition and phosphorylation of these by guest on March 24, 2020 http://www.jbc.org/ Downloaded from proteins. Antisera that specifically recognize the four 12-kDa P-proteins (P1, P2a, P2b, P3) were used to examine levels of these proteins in ribosomes isolated from a number of plant organs, including kernel tissues during seed maturation. Levels were also examined in ribosomes of root tips following flooding (anoxia), an environmental stress condition known to promote selective mRNA translation (28). We observed considerable developmentally and environmentally regulated heterogeneity in the levels and phosphorylation of these proteins.

Plant Material and Oxygen-Deprivation Treatment
Maize (Zea mays L.) (inbred B73, gift of Pioneer Hi-Bred International, Johnston, IA) plants were grown in the field, and leaves of ear husks, ears and silks were harvested at silk emergence. Ears were hand pollinated and harvested at 10, 15, 20, 25, 30 40 days postpollination (DPP) and after complete desiccation to isolate embryos (including the scutellum and embryonic axis), aleurone (tissue included the aleurone layer and attached pericarp) and endosperm (refers to tissue within the pericarp and aleurone, excluding the embryo).
For seedling tissue, kernels were surface-sterilized with 0.25% (v/v) sodium hypochlorite, imbibed for 8 h and germinated in the dark for 4 to 5 days at room temperature.
Previously described methods were used for oxygen deprivation (anoxia) of intact seedlings by submergence in an aqueous solution that was continuously sparged with 99.995% argon (28).
The apical 1 cm of the primary root and the entire coleoptile were harvested. All samples were frozen directly in liquid N 2 and stored at -80 o C.

RNA Isolation and Northern Hybridization
Total RNA was extracted following a CsCl-gradient method (29) and RNA blots (20 µg RNA per sample) were prepared (30)

Isolation of Ribosomes and Soluble Proteins and Purification of Acidic Ribosomal Proteins
One to 10g of tissue was ground to a fine powder under liquid N 2 with a mortar and pestle, and hydrated in 2 to 20 ml extraction buffer A (0. Acidic ribosomal proteins were purified from coleoptile ribosomes as previously described by Bailey-Serres et al. (26) in the absence of sodium molybdate.

Isolation of Soluble and Membrane-bound Ribosomes
Ribosomes were isolated following the protocol outlined by Mösinger and Schopfer (32).
One to 10g of tissue was ground to a fine powder under liquid N 2 with a mortar and pestle, and

Production of Polyclonal Antibodies
Peptides specific to maize P-proteins (A 44 LFAKLLEKRNVED 57 for P1; E 40 LLLSQLSGKD 50 for P2a; L 39 EFLLTELKDKDI 51 for P2b; and R 9 NNGGEWTAKQHSGEI 24 for P3) were synthesized, conjugated with a carrier protein (KLH, keyhole limpet hemocyanin for P2a, P2b and P3; tetanus toxoid for P1) and injected into rabbits. Antisera against P2a and P3 were purified by affinity chromatography using the specific peptide bound to a sepharose column (Quality Controlled Biochemicals Inc., Brighton, MA).

Maize P-protein Transcript Accumulation is Developmentally Regulated
We examined the abundance of P1, P2a, P2b and P3 mRNA transcripts in total RNA from several organs of maize and in kernel tissues during seed maturation and found that the accumulation of individual P-protein transcripts is developmentally regulated (Figure 1). Among the organs and tissues examined, transcript accumulation for all four 12-kDa P-proteins was highest in coleoptiles and immature ears, relative to 18S rRNA levels, as expected for organs undergoing rapid cell division and differentiation (33). P1, P2a and P3 mRNA accumulation patterns were very similar. These transcripts were detected at very low levels in leaf, silk and pollen, where, by contrast, P2b transcripts accumulated to moderately high levels. In all of the kernel tissues examined, P1, P2a and P3 mRNAs were abundant, with the highest levels at 15 DPP, whereas P2b mRNA was present at low levels. This variation in mRNA accumulation led us to examine the possibility of developmental differences in P-protein composition of ribosomes.

Specific Antisera Against the 12-kDa P-proteins of Maize Detect Phosphorylation Variants and Protein Complexes
Peptides specific to each of the four types of 12-kDa P-proteins of maize were synthesized and used to prepare antisera in rabbits. To evaluate the specificity of the antisera, ribosomes were isolated from coleoptiles, washed with high salt (0.8 M NH 4 Cl/50% ethanol) under conditions which release the 12-kDa P-proteins (26), and immunoblot analyses were performed. Pre-immune sera showed no detectable cross-reaction to r-proteins (data not shown). P1, P2a, P2b and P3 antisera detected polypeptides of distinct molecular mass from coleoptile ribosomes, confirming that the antisera were specific ( Maize P1 is encoded by a single-copy gene, has a predicted molecular mass of 11.0 kDa, and three putative phosphorylation sites (26,27). P1 antisera recognized polypeptides with apparent molecular masses of ~14.5 and 50 kDa in coleoptile ribosomes ( Maize P2a is encoded by a gene family of approximately four members, has a predicted molecular mass of 11.5 kDa, and five to six putative phosphorylation sites (26,27). The P2a antiserum detected a group of ~12 kDa polypeptides in coleoptile ribosomes ( Figure 2A, P2a panel, lane 1). The detection of multiple 12 kDa forms of P2a is most likely is due to the expression of more than one rpp2a gene. The P2a antiserum also detected groups of ~26, ~42-48, and ~60 kDa polypeptides that were not detected with the other antisera. Following elution from ribosomes with high salt these polypeptides had slightly altered electrophoretic mobility ( Figure 2A, lane 3, white, gray and stippled arrows, respectively). However, not all of the 26-kDa form was released from the salt-washed ribosomes (lane 2). We were unable to promote or inhibit formation of the P2a complexes in ribosomes or eluate samples by manipulation of SDS, urea, sulfhydryl reducing agents, or heat, indicating that they involve strong hydrophobic interactions (data not shown). These results suggest that P2a is present in dimeric (24)(25)(26) and additional multimeric complexes (42-48 kDa, 60 kDa) in coleoptile ribosomes. Maize P2b is encoded by a single gene, has a predicted molecular weight of 11.8 kDa, and three putative phosphorylation sites (26,27). P2b was detected as a 14-kDa polypeptide in coleoptile ribosomes ( Maize P3 is encoded by one to two genes, has a predicted molecular mass of 12.2 kDa, and three putative phosphorylation sites (26,27). The P3 antiserum detected a 15-kDa polypeptide in coleoptile ribosomes ( (Figure 2A). More slowly migrating forms of all of the P-proteins were observed following extraction from ribosomes with high salt. We determined that the in vivo phosphorylation status of these proteins was maintained if ribosomes were isolated in the presence of the nonspecific phosphatase and kinase inhibitor sodium molybdate. The effect of the presence or absence of sodium molybdate in the extraction and resuspension buffers is shown for root tip ribosomes in Figure 2B. To confirm loading of similar quantities of r-proteins in each sample, immunoblots were co-incubated with an antiserum prepared against r-protein S6 (30 kDa). In all cases, the presence of sodium molybdate increased the level of the more slowly migrating forms of the different P-proteins ( Figure 2B). A panel for P2b is not shown since this form was not detected in root-tip ribosomes (see Figure 3). These results indicate that root extracts contain a phosphatase activity that is inhibited by sodium molybdate and that P1, P2a and P3 of root ribosomes are predominantly phosphorylated. By contrast, the P-proteins of coleoptile ribosomes had faster electrophoretic mobility ( Figure 3, compare root tip and coleoptile lanes) indicating that these proteins are either dephosphorylated in coleoptile ribosomes or a phosphatase was not inhibited during the extraction. The ability of a ribosomeassociated kinase to phosphorylate coleoptile P-proteins was evidenced by the change in electrophoretic mobility following resuspension in a buffer lacking sodium molybdate ( Figure   2A, panel P3) or release from ribosomes with high salt (panels P1, P2a and P2b). These results suggest that the phosphorylation status of these proteins may be regulated by ribosomeassociated kinases.

12-kDa P-protein Phosphorylation is Reduced in Response to Anoxia
Our previous investigations indicated that the phosphorylation status of the 12-kDa Pproteins is altered in response to anoxia (26). The immunoblot shown in Figure 2C demonstrates that when intact seedlings were deprived of oxygen for up to 24 h, an increase was observed in the amount of the faster migrating, dephosphorylated forms of P1, P2a and P3. This result unambiguously demonstrates that 12-kDa P-proteins are dephosphorylated under anoxia.

Developmental Distinctions in the 12-kDa P-protein Composition of Ribosomes
Levels of the 12-kDa P-proteins in ribosomes from several organs (root tip, coleoptile, leaf, silk and ear) and kernel tissues (embryo, aleurone and endosperm) were surveyed relative to levels of r-protein S6. Ribosomes were isolated in the presence of sodium molybdate to control protein phosphorylation status during extraction. The immunoblots shown in Figure 3 demonstrate considerable developmental differences in quantity and electrophoretic mobility of the 12-kDa P-proteins relative to the level of S6 (shown in P2 panel) in ribosomes. was only detected as a monomer in these kernel samples. As seen for P1, the apparent molecular mass of P2a in root ribosomes was higher than that of coleoptiles. The apparent molecular mass of P2a in leaf, silk and ear ribosomes was even higher than that of root tips. The observed variations in electrophoretic mobilty could reflect differential expression of the rpp2a genes and/or distinctions in protein phosphorylation.
P2b was detected at dramatically different levels in ribosomes isolated from various organs and tissues (Figure 3, P2b panel). A 14-kDa form of P2b was detected in coleoptile ribosomes, whereas a 14.5-kDa form was detected in leaf, silk and 30 DPP aleurone ribosomes.
P2b was not detected in root tip ribosomes and was present at very low levels in ear, embryo and endosperm ribosomes. Higher levels of P2b in coleoptile, leaf and silk ribosomes correlated with the mRNA accumulation data (Figure 1). P2b levels were low to undetectable in ear and root tip ribosomes, despite the detection of P2b mRNA in these organs, whereas P2b levels in aleurone ribosomes were higher than predicted from the RNA blot data. The faster migration of P2b of coleoptile ribosomes was consistent with that observed for the other P-proteins.
The lack of variability in the abundance of P3 associated with ribosomes was in marked contrast to that observed for the two forms of P2. P3 was detected as a 15.5-kDa polypeptide at similar levels in all ribosome samples, with the exception of coleoptile ribosomes where it was detected as a 15-kDa polypeptide, evidently due to reduced phosphorylation (Figure 3, P3 panel).
A small amount of dephosphorylated P3 was consistently detected in root tip ribosomes. The  (Figure 1).

Regulation of 12-kDa P-protein Levels Occurs during Kernel Development
Given the considerable developmental regulation in accumulation of the 12-kDa proteins in ribosomes in the maize organs and tissues surveyed, we decided to monitor P-protein levels during the temporal development and maturation of the kernel. Figure 4A-C compares the Pprotein content of ribosomes over the time course of kernel development and maturation, relative to levels of r-protein S6. The maize embryo consists of the embryonic axis surrounded by the scutellum, a modified cotyledon. In embryos monitored from 15 to 40 DPP, levels of P1, P2a and P3 were not dramatically altered ( Figure 4A). P1 was detected as a 15-kDa polypeptide, P2a was detected only in the 12.5-kDa monomeric form, and P3 was detected as a 15.5-kDa polypeptide. By contrast, levels of P2b increased dramatically in embryo ribosomes after 25 DPP. All four P-proteins were detected in ribosomes isolated from dry embryos of mature kernels, indicating that ribosomes stored in the seed embryo possess these proteins. There was no indication that phosphorylation status was modulated during embryo maturation.
The kernel aleurone, the outermost cell layer of the endosperm, develops until about 40 DPP, at which time it has become quiescent and desiccated; upon seed imbibition the stored ribosomes actively synthesize the starch hydrolases required to mobilize nutrients. Levels of P1, P2a, P2b and P3 in ribosomes increased during early aleurone development (10 and 15 DPP) and again at the late maturation stage (30 and 40 DPP) ( Figure 4B). An increase in the electrophoretic mobility of P2a that was consistent with dephosphorylation was observed at 40 DPP. The level of P2b was less dramatically modulated in the aleurone than in the kernel embryo or endosperm.
The endosperm is the triploid, nutritive organ of the kernel that stores carbohydrate, lipid and protein reserves for the embryo. The endosperm develops from fertilization until 12 to 15 DPP, at which time grain-filling begins and proceeds until approximately 40 to 50 DPP, when a spatial progression in programmed cell death occurs (34). We observed that levels of P1, P2a and P3 were not markedly regulated during endosperm development ( Figure 4C). By contrast, levels of P2b increased dramatically after 25 DPP, as observed in embryo ribosomes. Ribosomes could not be isolated in sufficient quantities from dry endosperm to perform immunoblot analyses, most likely due to a reduction in ribosome levels at the end of endosperm maturation (data not shown).  (37,38). Cell fractionation was performed to examine the 12-kDa P-proteins of soluble and membrane-associated ribosomes of 40 DPP endosperm and coleoptile. Cell extracts were prepared in the absence of detergent and centrifuged to produce a supernatant that contained soluble ribosomes and a pellet that contained membrane-associated ribosomes. The pellet was resuspended in a detergent-containing buffer and re-centrifuged to obtain a clarified supernatant of detergent-solubilized membrane-associated ribosomes. In the endosperm sample some of the ribosomes were not released by this detergent treatment and were re-pelleted. This pellet contained high levels of zeins and r-proteins, confirming the purification of a fraction enriched in PB-associated ribosomes ( Figure 6, γ−zein panel). A similar detergent-resistant fraction was not obtained from coleoptiles. The P-proteins levels in soluble and membrane-associated ribosomes, relative to r-protein S6, were not identical in coleoptile or endosperm ( Figure 6). In coleoptiles, slightly reduced levels of these proteins were reproducibly detected in membrane-associated ribosomes. This was especially evident for the P2a monomer ( Figure 6) and dimer (data not shown). In 40 DPP endosperm, the presence and abundance of P1, P2a and P3 showed little variation between soluble, membrane ribosomes and PB-associated ribosomes. In contrast, P2b levels were reduced in membrane-associated ribosomes compared to soluble ribosomes and P2b was undetectable in PB-associated ribosomes.

Distinctions in Ribosomes Associated with Storage Protein Bodies of Endosperm
These results provide evidence that the 12-kDa P-protein composition of ribosomes surrounding PBs and translating zein mRNAs is distinct from ribosomes translating soluble proteins.

Heterogeneity in Ribosomal P-protein Composition
The results presented here clearly demonstrate that maize ribosomal protein composition is variable with respect to the four ~12 kDa P-proteins, P1, P2a, P2b and P3. Ribosome heterogeneity is due to differences in, (1) phosphorylation of the 12 kDa P-proteins, (2) the presence and abundance of distinct 12-kDa P-proteins at the tissue and subcellular (membrane vs. soluble) levels, and (3) the presence and abundance of multimeric complexes of P2a.

Phosphorylation of the P-proteins of Root Ribosomes is Developmentally and Environmentally Regulated
Our previous studies indicated that the maize 12 kDa P-proteins are phosphoproteins and that their phosphorylation is modulated in response to anoxia (26). Our current analyses reveal several examples of modulation of phosphorylation of the 12 kDa P-proteins in response to cues from the environment and during development. The isolation of ribosomes from root tips in the presence of sodium molybdate, a non-specific phosphatase and kinase inhibitor, revealed that the phosphorylation of P1, P2a and P3 (P2b was not detected in root tip ribosomes) was reduced in response to anoxia, consistent with earlier predictions. P1, P2 and P3 of coleoptiles migrated at a rate similar to the dephosphorylated forms of root ribosomes, suggesting that these proteins are either dephosphorylated in coleoptiles or a phosphatase was not inhibited during extraction.
Also, the phosphorylation status of P2a was dephosphorylated in late aleurone development.
Several kinases that phosphorylate the P-proteins have been isolated from yeast, however very little is known about the phosphatases that act on these proteins. Recently, a protein phosphatase, isolated from a ribosome-free extract from yeast, was shown to dephosphorylate P1, P2 and P0 in vitro (39). Protein kinase 60S (PK60S) (40) casein kinase II (CK II) (41,42), ribosome acidic protein kinase I (RAP I) (43), and RAP II (44) have been shown to in vitro phosphorylate P-proteins from several organisms. In yeast, Bou et al. (44) demonstrated that RAP II and PK60S preferentially phosphorylate P1β and P2α, whereas RAP I and CK II modify all of the P-proteins. The distinct phosphorylating activities of the various P-protein kinases suggest that the differential phosphorylation of the P-proteins is of functional significance. Since all of the maize P-proteins were modified during anoxia in the same manner, we predict that this modulation is due to the inactivation of a general P-protein kinase and/or activation of a general P-protein phosphatase. In contrast to the global P-protein dephosphorylation observed in response to anoxia, the developmentally regulated dephosphorylation observed in late aleurone maturation was P2a specific. This modulation could be due to the inactivation of a kinase and/or activation of a phosphatase that specifically modifies P2a.
Yeast strains in which the phosphorylated C-terminal serine of P1α was mutated to an alanine were less sensitive to osmotic stress than wildtype or mutant strains in which this serine was mutated to threonine, leading to the prediction that dephosphorylation of P1 is required for an adaptive response to stress (25). Dephosphorylation is plausibly a global down-regulator of protein synthesis since dephosphorylation of the C-terminal serine of rat P2 reduced translational activity in vitro (17). Additional studies are needed to determine if the dephosphorylation of P1, P2a and P3 in response to anoxia in maize is involved in some aspect of the translational control observed under this stress.

Ribosome Heterogeneity is Due Mainly to Variations in P2
Examination of the ribosomal P-proteins in a range of organs revealed striking heterogeneity, especially in P2 composition. The novel plant P-protein, P3, showed little variation in accumulation and was less efficiently eluted from salt-washed ribosomes than the other stalk proteins. P1 accumulation patterns were slightly more complex. P1 was detected as a monomer with varying electrophoretic mobility. P1 was less efficiently released from ribosomes with high salt than P2a or P2b. By contrast, considerable tissue-specific and developmental regulation was observed in the levels of the two forms of P2. P2a levels were higher in root tip, coleoptile, embryo and endosperm ribosomes as compared to leaf, silk, immature ear and kernel aleurone ribosomes. The low levels of P2a were reciprocated by higher accumulation of P2b in ribosomes of leaf, silk and aleurone, but apparently not in the ear at silk emergence, or aleurone during early development (10 DPP).
Variations in P2 also included the accumulation of P2a complexes (dimers, and other multimeric forms). P2a dimers were observed in all samples except in ribosomes of kernel tissues. The N-terminal regions of the yeast 12-kDa P-proteins are predicted to form α-helices with hydrophobic residues spaced at intervals that would promote coiled-coil interactions (45).
Similar to yeast, maize P2a is predicted to form an α-helix with a strong hydrophobic edge that may result in the observed dimers and multimers (K.S.M. and J.B.S., unpublished results). The absence of P2a dimers in kernel tissue ribosomes is enigmatic, but may reflect differences in phosphorylation at N-terminal sites or differential expression of P2a gene family members.
The most striking distinctions we observed were in the levels of P2b. This protein was not detected in ribosomes from roots and showed a dramatic developmentally programmed increase in embryo and endosperm. P2b was also undetectable in the PB-associated ribosomes from 40 DPP endosperm but was present in the soluble ribosomes from the same sample.
Although the functional significance of the observation is unclear, these distinctions could be involved in the translational control of zein protein synthesis if P2b containing ribosomes do not translate zein mRNAs.
Recently, Zurdo et al. (24) demonstrated in yeast that binding of P1 to the ribosome must proceed binding of P2, suggesting that assembly of the P-protein stalk is an ordered process. We observed that both P2a and P2b were efficiently eluted from ribosomes washed with high salt, whereas P1 and especially P3, were less efficiently eluted. This, together with the limited variation in the amounts of P1 and P3 suggests that P1 and P3 precede and may be required for P2 assembly. Given the low levels of P2 proteins in leaf, silk, ear and early aleurone ribosomes, the presence of P1 and P3 may be sufficient for translation. The extensive level of P2 modulation and their efficient removal from salt-washed ribosomes may indicate that these proteins are not necessary for protein synthesis but impart some mode of translational regulation.

Heterogeneity in P-protein Composition and its Possible Functional Significance
Our results illustrate clear distinctions in the presence, abundance and phosphorylation of Our results show that distinctions in the 12-kDa P-proteins of maize ribosomes are determined by development and environment. To account for our data and to consider the functional significance of ribosome heterogeneity on translational activity we present a model based on the structure depicted by Ballesta et al. (6,46). We propose that ribosomes with distinct 12-kDa P-proteins composition exist in maize (Figure 7). Ribosomes may possess a minimal acidic protein complex of P0, P1 and P3 (Ribosome Type I), or a minimal complex distinguished by quantitative differences in P2 composition (Ribosome Types II-V). Type I ribosomes predominate in aleurone at 10 DPP and ears at silk emergence. These ribosomes may have a basal level of eEF2-dependent GTPase activity and hence a basal rate of translational elongation.
Type II ribosomes contain the minimal complex with the addition of P2a and predominate in kernel tissues. Type III ribosomes contain the minimal complex and multimeric forms of P2a and predominate in roots and coleoptiles. Type IV ribosomes contain the minimal complex with the addition of P2b and predominate in leaf and silk. Finally, Type V ribosomes, possessing all four 12-kDa P-proteins, may exist in cells that express both P2a and P2b. Ribosomes possessing stalks with distinct P2 composition (Type II -IV), may have distinct levels of eEF2-dependent GTPase activity (higher or lower than the basal level), and/or differential efficiency in translation of specific mRNAs. Since P1, P2 and P3 phosphorylation levels are modulated in response to environmental stress (anoxia) and during development, we propose that C-terminal phosphorylation further regulates translational elongation. This ribosome heterogeneity, largely due to variations in P2 composition, strongly indicates a regulatory role of the acidic stalk of the large subunit in translation at the global or message specific level.      Soluble (S), membrane-associated (M) and γ−zein protein body-associated (PB) ribosomes isolated from coleoptiles or 40 DPP endosperm were resolved on SDS-PAGE gels, transferred to nitrocellulose and incubated simultaneously with either S6, P1, P2a, P2b, P3, or zein antisera.
The apparent molecular weight of each protein (kDa) is indicated on the right as determined by the migration of molecular weight markers.