Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1998 Jan;116(1):379–385.

Synthesis and Phosphorylation of Maize Acidic Ribosomal Proteins1

Implications in Translational Regulation

Raúl Aguilar 1, Leonel Montoya 1, Estela Sánchez de Jiménez 1,*
PMCID: PMC35179

Abstract

The objective of this research was to determine the role of acidic ribosomal protein (ARP) phosphorylation in translation. Ribosomes (Rbs) from germinated maize (Zea mays L.) axes had four ARP bands within 4.2 to 4.5 isoelectric points when analyzed by isoelectric focusing. Two of these bands disappeared after alkaline phosphatase hydrolysis. During germination a progressive change from nonphosphorylated (0 h) to phosphorylated ARP (24 h) forms was observed in the Rbs; a free cytoplasmic pool of nonphosphorylated ARPs was also identified by immunoblot and isoelectric focusing experiments. De novo ARP synthesis initiated very slowly early in germination, whereas ARP phosphorylation occurred rapidly within this period. ARP-phosphorylated versus ARP-nonphosphorylated Rbs were tested in an in vitro reticulocyte lysate translation system. Greater in vitro mRNA translation rates were demonstrated for the ARP-phosphorylated Rbs than for the non-ARP-phosphorylated ones. Rapamycin application to maize axes strongly inhibited S6 ribosomal protein phosphorylation, but did not interfere with the ARP phosphorylation reaction. We conclude that ARP phosphorylation does not depend on ARP synthesis or on ARP assembly into Rbs. Rather, this process seems to be part of a translational regulation mechanism.

A distinctive characteristic of eukaryotic Rbs is the phosphorylation status of their ARPs (Hershey, 1989). Studies on ARPs from different eukaryotes (Zinker and Warner, 1976; Shimmin et al., 1989; Wool et al., 1991) have demonstrated that these proteins are conserved through evolution, particularly at the carboxy-terminal end (Remacha et al., 1995b). They have been classified into two groups, namely P1 and P2 (Wool et al., 1991). These proteins are located in the stalk of the large ribosomal subunit (Strycharz et al., 1978) and are known to participate in translation by interacting with translation elongation factors (Sánchez-Madrid et al., 1979; MacConnell and Kaplan, 1982).

Assembly of ARPs in the Rb occurs in the cell cytoplasm, where ARPs constitute a free protein pool (Mitsui et al., 1988; Saenz-Robles et al., 1990). Studies regarding ARP gene identification have reported the presence of two genes for these proteins in mammals (Wool et al., 1991). Lower eukaryotes, however, have more ARP; four have been reported in yeast (Remacha et al., 1990; Beltrame and Bianchi, 1990) and even eight have been reported for Trypanosoma cruzi (Vázquez et al., 1992). In plants two different P-protein genes have been found for rice (Goddemeier et al., 1996) and three for maize (Zea mays L.) (Bailey-Serres et al., 1997). The plant P proteins showed homology to the carboxy-terminal ends of their animal counterparts (Ballesta and Remacha, 1996).

The expression of these proteins in yeast has been demonstrated to be at least partially autoregulated by the pool size of the reciprocal isoforms (Bermejo et al., 1994). However, the mechanism that regulates ARP assembly and/or exchange within the Rb is not fully understood. For some time it was thought that ARP phosphorylation played a relevant role in the stability of ARP-Rb association (Naranda and Ballesta, 1991). However, this role was not further supported by in vivo evidence showing that ARP assembled into Rbs in yeast mutants lacking the target phosphorylable Ser residue (Ballesta and Remacha, 1996).

Seed embryonic axes reinitiate protein synthesis at the beginning of germination, based primarily on stored mRNA and preformed Rbs. In maize seeds ribosomal protein synthesis has been demonstrated to occur early in germination (Beltrán et al., 1995). However, precise information regarding de novo ARP synthesis and/or ARP assembly into Rbs during this period is not available at present.

Previous work from our laboratory has shown that maize Rbs contain two ARPs similar to the mammalian ribosomal proteins P1 and P2, which actively incorporate 32P-orthophosphate during germination in a tightly regulated manner (Pérez-Méndez et al., 1993). However, it is not known whether ARP phosphorylation has a relevant role in regulating translation within this period. The present research focuses on the course of ARP synthesis and phosphorylation in maize embryonic axes during germination and evaluates the phosphorylation role ARPs in Rb assembly and translation.

MATERIALS AND METHODS

Biological Material

Maize (Zea mays L. var. Chalqueño) embryonic axes were obtained by manual dissection and disinfected as reported previously (Pérez-Méndez et al., 1993). The axes were incubated for different periods under sterile conditions on Murashige and Skoog medium (Murashige and Skoog, 1962) in the dark at 25°C. Specific experimental conditions are described in more detail below.

ARP Isolation

Rbs were isolated from axes according to the method of Scharf and Nover (1982), with modifications as follows: the axes were homogenized to a fine powder in liquid N2 and resuspended in 10 volumes of extraction buffer A1 (20 mm Tris-HCl, pH 7.8, 5 mm MgCl2, 20 mm KCl, 1 mm NaF, and 0.5% β-glycerophosphate). The homogenate was centrifuged at 27,000g for 30 min, and then at 250,000g through a Suc cushion (0.5 m Suc and 0.5 m KCl in buffer A1) for 3.5 h. The ribosomal pellet was resuspended (20 mg/mL) in buffer containing 10 mm Tris-HCl, pH 7.4, 12 mm MgCl2, 80 mm KCl, and 5 mm β-mercaptoethanol plus 1 volume of ammonium buffer (1.5 m NH4Cl, 20 mm MgCl2, 3 mm β-mercaptoethanol, and 20 mm Tris-HCl, pH 7.4) and stirred for 20 min in an ice bath. Ethanol was added slowly until a 1:1 (v/v) mixture was obtained. The mixture was stirred for 20 min more in the ice bath and centrifuged at 10,000g. The pellet was discarded and ARPs were precipitated from the supernatant with 2.5 volumes of acetone at −20°C overnight. Further purification of ARPs was achieved by carboximethyl-cellulose treatment, as described by Juan-Vidales et al. (1981). Proteins were measured by the Bradford technique (Bradford, 1976) and BSA (Sigma) was used as a standard.

Similarly, Rbs obtained from rat liver (Petermann, 1971) were the source for ARP isolation, following the same procedure. These proteins were used to raise rabbit antibodies by a conventional protocol.

Cytoplasmic ARPs

The postribosomal supernatant from the axes homogenate after the 250,000g centrifugation was precipitated with 5 volumes of acetone at −20°C, allowed to stand overnight, and centrifuged at 5,000g. After the acetone evaporated, the pellet was ethanol-ammonium extracted, as indicated in the procedure for ARP isolation. ARPs were resuspended in buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, and 0.05% Nonidet P-40) and immunoprecipitated with rat liver ARP antibodies (1:1,500) as previously reported (Sánchez de Jiménez et al., 1997), except that the Sepharose A-IgG ARP complex was eluted with 9.5 m urea.

Polyacrylamide Gel IEF of Ribosomal Proteins

To analyze ARPs by IEF, whole Rbs (2.5–5.0 OD260) were placed on 5% polyacrylamide, 8 m urea gels, and 2.5 to 5.0 pH ampholytes (Sigma) for 18 h in a vertical slab-gel unit from Hoefer (San Francisco, CA) (Juan-Vidales et al., 1984). Protein bands were visualized by the silver-stain method.

PAGE of ARPs

ARPs were purified from maize axes by Rb ethanol-ammonium extraction and carboxy-methyl cellulose chromatography (Pérez-Méndez et al., 1993). ARPs were resolved by one-dimensional SDS-PAGE following the method of Laemmli (1970) in 12% acrylamide gels. Proteins were visualized by Coomassie blue R-250.

Alkaline-Phosphatase Treatment of Rbs

Isolated Rbs (40–50 μg) from embryonic axes of 24-h germinated seeds were resuspended in 90 μL of 20 mm Hepes-KOH, pH 7.6, 5 mm magnesium acetate, 125 mm potassium acetate, and 6 mm β-mercaptoethanol buffer; 1.5 units of alkaline-phosphatase Sepharose beads (Sigma) were added. Reaction mixtures were incubated at 37°C for 60 min, and then centrifuged (12,000g for 20 min) to remove insoluble Sepharose. The supernatant was analyzed by IEF.

Rb Autophosphorylation

Rbs (approximately 5.0 OD260) from ungerminated maize axes were incubated in 250 mm Hepes-KOH buffer, pH 7.6, containing 0.65 mm β-mercaptoethanol, 0.925 mm magnesium acetate, 0.8 mm ATP, and 80 mm β-glycerophosphate in a final volume of 150 μL, according to Sepúlveda et al. (1995). After incubation at 27°C for 30 min, the reaction mixture was centrifuged at 250,000g for 1 h and the Rbs were analyzed by IEF.

Slot-Blot Analysis

Ten micrograms of ARPs or 120 μg of total ribosomal proteins purified by the method of Ramjoué and Gordon (1977) was applied to nitrocellulose sheets (Schleicher & Shuell) and tested according to Memelink et al. (1994) against either yeast (1:50) (kindly donated by Dr. J.P.G. Ballesta, Universidad Autónoma de Madrid, Spain) or rat liver ARP antibodies (prepared in this lab; 1:1500). Purified goat anti-rabbit IgG conjugated to horseradish peroxidase (1:1000, GIBCO-BRL) was used as a second antibody. Color was developed with 4-chloro-1-naphtol substrate plus 30% (v/v) H2O2.

In Vivo Synthesis of ARPs

Embryonic axes (400 mg) were incubated for either 4 or 14 h at 25°C, and pulse labeled with 100 μCi of [35S]Met (specific activity 1155 Ci mmol−1, DuPont NEN) for the last hour of incubation in a final volume of 1 mL. A control group of 14-h incubated axes was also prepared but in the presence of α-amanitin (12 μg/mL) (Sigma). The axes were then rinsed three times with distilled water and frozen in liquid N2 until used. The Rbs were isolated from the axes as indicated, and the ARPs were extracted and resolved by IEF. Slab gels were treated for fluorography by soaking in amplifier solution (Amersham) for 20 min, dried under a vacuum in a gel dryer (Bio-Rad), and exposed to Hyper film (Amersham) for 2 weeks. Alternatively, the gels were cut at the ARP bands and counted in a liquid-scintilation counter (Packard, Meriden, CT).

In Vitro Translation System

A reticulocyte lysate translation kit (Boehringer Mannheim) was used following the manufacturer's standard assay. The lysate was depleted of its Rbs by ultracentrifugation (Sánchez de Jiménez et al., 1997). Ten microliters of Rb-depleted retitulocyte lysate was supplemented with maize axes Rbs (0.3–0.5 OD260) from either 0- or 18-h incubated axes in or not in the presence of 0.1 μm rapamycin for the last 2 h. Twenty micrograms of total RNA and 50 μCi [35S]Met (specific activity 1140 Ci mmol−1, DuPont NEN) were added to the system in a final volume of 25 μL. Mixtures were incubated at 30°C and aliquots (2 μL) were taken every 5 min for 25 min. The samples were precipitated with 10% (w/v) TCA containing 2% (w/v) casein hydrolysate (Sigma), kept for 10 min on ice, boiled for 15 min, cooled on ice, and filtered through GF/C filter paper (Whatman). The filters were washed four times with 5% TCA containing 0.1% casein hydrolysate, dried, and counted in a liquid-scintillation counter (Packard).

RESULTS

Characterization of Maize Axes ARPs

Rbs isolated from germinated maize axes were analyzed by IEF on a 2.5 to 5.0 pH range. The silver nitrate-stained gel showed four ARP bands of pI values ranging from 4.2 to 4.5 (Fig. 1A, IEF). Rbs previously treated with alkaline phosphatase and then analyzed by this procedure showed only two bands, the upper, less-acidic bands (Fig. 1B, IEF). As a control, maize ARPs purified from these Rbs were analyzed by SDS-PAGE. Two bands of 14.5 and 16 kD were observed (Fig. 1C, SDS-PAGE), as previously reported (Pérez-Méndez, et al., 1993). These data indicate that germinated maize axes contain two ARP peptides (P1 and P2) present as phosphorylated and nonphosphorylated forms (Fig. 1).

Figure 1.

Figure 1

Open in a new tab

Rbs (40 μg) from 24-h germinated maize axes were analyzed by 2.5 to 5.0 pH IEF and stained with silver nitrate (A) or incubated with alkaline-phosphatase (1.5 units) at 37°C for 25 min and then electrofocused and silver stained as above (B). Letters indicate the IEF position of the nonphosphorylated (a and b) or phosphorylated (c and d) ARPs. ARP (20 μg) purified from 24-h germinated axes as a control (C) and molecular markers (5 μg) (MM) were gel electrophoresed and stained with Coomassie blue. Numbers indicate positions of the molecular mass markers (in kilodaltons).

ARPs from maize Rbs at different germination periods, 0, 4, 14, and 24 h, were IEF analyzed. A changing IEF pattern was observed within this period in the stained gels. ARPs from ungerminated axes (0 h) showed only the two nonphosphorylated ARP bands with high pIs (Fig. 2, a and b bands). As germination progressed, the other two bands with lower pIs (Fig. 2, c and d bands) appeared to increase in intensity, whereas bands a and b decreased in intensity, suggesting changes in the phosphorylated/dephosphorylated ARP ratio during germination. The total amount of ARPs per OD260 ribosomal unit, however, remained similar through this period. Rbs from ungerminated axes (nonphosphorylated ARPs) were phosphorylated in vitro. Results showed phosphorylated ARP forms in these Rbs, similar to the pattern for Rbs from 24-h germinated axes (Fig. 2, O–P).

Figure 2.

Figure 2

Open in a new tab

IEF analysis of ARPs from germinating seed axes. Rbs from axes germinated for 0, 4, 14, or 24 h, and for 0 h after 30 min of autophosphorylation (0-P) were obtained as indicated in Methods. Rbs (40–45 μg) from each experimental set were resolved by IEF using ampholines, 2.5 to 5.0 pH range, and silver nitrate stained to resolve their ARP content. Letters indicate the IEF position of the ARPs as above.

The presence of free ARPs in the cytoplasm of the axes was investigated in the postribosomal supernatants of ungerminated and germinated axes. The acidic ethanol-ammonium-extracted proteins from these supernatants were tested by slot-blot analysis with either yeast or rat liver ARP antibodies. Purified ARPs from 24-h germinated axes were included as a control (Fig. 3, Rb). A positive cross-reaction was observed in the ethanol-ammonium-extracted cytoplasmic proteins with either of the antibodies tested (Fig. 3A). Further analysis of these proteins by IEF revealed only two bands of ARPs with high pIs corresponding to the nonphosphorylated ARP forms (Fig. 3B, a and b bands). These results indicate the presence of a cytoplasmic, nonphosphorylated ARP free pool in maize axes for both ungerminated and germinated seeds.

Figure 3.

Figure 3

Open in a new tab

Either acidic cytoplasmic proteins isolated from 24-h germinated axes, as indicated in Methods (Sn), or their ribosomal proteins (Rb) (control) were used for these experiments. A, Ten micrograms of each protein group was applied to the nitrocellulose membrane and tested by slot-blot analysis using rat ARP antibodies (diluted 1:1500). The second antibody was rabbit IgG conjugated to peroxidase (diluted 1:1000). B, The same protein samples were also analyzed by IEF and silver stained. Letters indicate the IEF position of the ARPs as above. The experiments were reproduced with cytoplasmic acid proteins from 0-h axes instead of the 24-h set, with very similar results.

De Novo Synthesis of ARPs during Germination

The next experiment was to investigate whether the cytoplasmic ARP pool from ungerminated axes was the ARP source for Rb assembly early in germination or if de novo ARP synthesis occurred within this period. Maize axes from 4, 8, and 14 h of germination were pulse labeled with [35S]Met. Ribosomal and cytoplasmic ARP [35S]-incorporation was determined either by IEF and fluorography (ribosomal ARPs) or by immunoprecipitation, elution, and direct sample counting (cytoplasmic ARPs). Results showed almost no [35S]Met incorporation (or low, even after long periods of film exposure) at early periods of germination (4 and 8 h) in ribosomal ARPs, whereas at 14 h of germination [35S]Met incorporation was already clearly observed in the ARPs (Fig. 4). On the other hand, the cytoplasmic ARPs showed a similar pattern of synthesis, with slow [35S]Met incorporation at early germination periods (4 and 8 h) but greater incorporation after 14 h (Table I). Transcription inhibition with α-amanitin during germination showed decreased [35S]Met incorporation into ARPs but not total inhibition at 14 h of germination (Fig. 4, 14α), suggesting the presence of stored ARP mRNAs in the ungerminated axes. This interpretation is further supported by recent experiments showing P2 mRNA among the stored mRNAs by northern-blot analysis using a homologous maize P2 cDNA probe (data not shown). Later, new mRNA transcription seems to occur.

Figure 4.

Figure 4

Open in a new tab

Maize embryonic axes incubated in Murashige and Skoog medium were pulse labeled with 100 μCi of [35S]Met during the last hour of incubation. The Rbs were isolated and ARPs were analyzed by IEF in a pH range of 2.5 and 5.0. The gel was prepared for fluorography as described in Methods. Approximately 10,000 cpm were loaded per well. The lanes correspond to: axes incubated for 4 or 14 h of germination (4 and 14, respectively), and axes allowed to imbibe with α-amanitin (12 μg/mL) then incubated for 14 h and [35S]-labeled as above (14α). The α-amanitin incubating conditions used here ensure large mRNA transcription inhibition (<5% of total RNA remaining transcription) (Beltrán et al., 1995). Arrows point to the position of the labeled bands. At 8 h the labeled pattern was like the 4-h pattern. These experiments were reproduced at least twice, with similar results.

Table I.

Synthesis of cytoplasmic ARPs: [35S]Met incorporation

Germination Period ARPs
h cpm mg−1 × 10−3
4 16.6
8 40.1
14 108.4
Open in a new tab

Maize axes (400 mg) were incubated for the stated periods and [35S]Met (100 μCi) was added during the last hour of incubation (see Methods). The cytoplasmic ARPs were immunoprecipitated and counted in a scintillation counter.

Relevance of ARP Phosphorylation on the Translation Process

The role of ARP phosphorylation in translation was analyzed in an in vitro translation system. This system was based on Rb-devoid reticulocyte lysate supplemented with either Rbs from 0- or 24-h-germinated maize axes and total maize axes RNA as a source of mRNA. The translation rate was measured as the amount of [35S]Met incorporated per minute per milligram of protein. About 2-fold more [35S]Met incorporation was found after 30 min of incubation in the translation system containing Rbs from 24-h germinated axes than in the one with ungerminated axes (0 h) (i.e. 24,350 versus 11,891 cpm/μg protein, respectively), suggesting a positive correlation between translation efficiency and phosphorylated ARPs in the Rbs.

A previous report from our laboratory (Pérez-Méndez et al., 1993) showed that S6 ribosomal protein of maize axes was phosphorylated during germination. Phosphorylation of this protein has been reported to stimulate translation of specific mRNAs, the 5′TOP (terminal oligopyrimidine) mRNAs, (Jefferies et al., 1994; Terada et al., 1994), as well as some Cap-dependent maize mRNAs (Sánchez de Jiménez et al., 1997). Therefore, a translation experiment was performed comprising S6-nonphosphorylated, and ARP-phosphorylated Rbs to confirm the role of ARP phosphorylation in translation. Rapamycin, a strong, specific inhibitor of pp70S6k, the enzyme responsible for in vitro phosphorylation of S6 RP (Price et al., 1992), was used for this purpose. Inhibition of S6 RP phosphorylation by rapamycin in maize axes has already been demonstrated (E. Sánchez de Jiménez, E. Beltrán-Peñá, and A. Ortíz-López, unpublished data), and a control experiment was also designed to demonstrate that this inhibitor has no effect on ARP phosphorylation. This was confirmed by Rb IEF analysis of rapamycin-exposed axes (data not shown). The in vitro-translation experiment was then performed with Rbs extracted from 18-h germinated maize axes that were previously rapamycin-inhibited or that had not been rapamycin-inhibited during the last 2 h of incubation and compared with the 0-h (nonphosphorylated) Rbs. The rate of protein synthesis measured with these Rbs showed faster [35S]Met incorporation into proteins in the 18-h Rb system than in the one with the Rbs from ungerminated axes (0 h), regardless of rapamycin application (Fig. 5). Calculation of the correspondent slopes showed mean values of 632 ± 53 for the 0-h Rbs versus 1434 ± 292 for the 18-h Rbs, indicating significant slower translation rates for the ARP-nonphosphorylated than the ARP-phosphorylated Rbs (P ≤ 0.01).

Figure 5.

Figure 5

Open in a new tab

An in vitro reticulocyte lysate translation system was used for testing maize Rb translation efficiency. Axes from 0 and 18 h of germination were treated or not treated with 0.1 μm rapamycin for the last 2 h of incubation, as indicated in Methods. The Rbs were isolated and used for translation. The translation system contained: 10 μL of Rb-depleted reticulocyte lysate, maize Rbs (0.3–0.5 OD260) either from ungerminated (nonphosphorylated ARPs) or germinated (phosphorylated ARPs) axes, 20 μg of total axes RNA, and 50 μCi of [35S]Met in a final volume of 25 μL. The mixture was incubated at 30°C. Every 5 min, 3-μL aliquots were taken and applied to the GF/C filter paper and washed. The dried membranes were counted and plotted versus time. These data are representative of at least three different experiments. A, Rbs from ungerminated (0 h) (○) or germinated (18 h) (□) axes. B, S6 phosphorylated rapamycin-inhibited Rbs from ungerminated (0 h) (•) or germinated (18 h) (▪) axes.

DISCUSSION

ARP interaction with elongation factors has been demonstrated to speed up protein synthesis in eukaryotic organisms (Sánchez-Madrid et al., 1981; Juan-Vidales et al., 1984). The lack of these proteins in yeast null mutants has been shown to result in very slow cell growth rates (Remacha et al., 1995a). However, the ARP phosphorylation role in the translation process of eukaryotic systems remains unclear. For some time it was thought that ARP phosphorylation was required for ARP exchange/assembly between the Rb and the cytoplasm (Sánchez-Madrid et al., 1981; Saenz et al., 1990). Later, evidence from in vivo site-directed mutation experiments in yeast showed normal ARP ribosomal content in the mutants lacking the target ARP phosphorylable Ser residue (Remacha et al., 1995b).

In the present case, the ARP content per Rb in the ungerminated axes was slightly lower than the one present in the germinated maize axes, although they differed greatly in their ARP phosphorylation status (Fig. 2). These data are in agreement with the notion that ARP phosphorylation is not necessary for Rb ARP assembly. Nevertheless, our results showed that ARP phosphorylation is a tightly regulated event during germination (Fig. 2) (Pérez-Méndez et al., 1993), and seems to be independent from ARP de novo synthesis. At 4 h of germination, Rbs showed little incorporation of [35S] label in their ARPs (Fig. 4), whereas, at this time, ARP phosphorylation in the Rbs is already occurring rapidly (Fig. 2) (Pérez-Méndez et al., 1993). However, it cannot be stated if ARP phosphorylation occurs after ARP has been assembled into the Rb or if it is a cytoplasmic event followed by rapid ARP exchange with the Rb unphosphorylated ARPs.

It is interesting to mention that a protein kinase specific for ARPs has been found tightly bound to the 60S subunit of maize axis Rbs (Sepúlveda et al., 1995), the subunit where ARPs are assembled. This ARP kinase has been shown to phosphorylate efficiently in vitro the ARPs already assembled in the Rb (Fig. 2B) (Sepúlveda et al., 1995), suggesting that the first proposition might be most probable. This is also supported by the finding of a nonphosphorylated cytoplasmic pool of ARPs in both the ungerminated and the germinated maize axes (Fig. 3). Moreover, [35S]-ARPs were observed mainly in the nonphosphorylated forms of the Rbs in the pulse-labeled experiment at 14 h of germination (Fig. 4), indicating that de novo-synthesized ARPs were incorporated into the Rbs in their nonphosphorylated form. Thus, the developmentally regulated ARP phosphorylation observed in the Rbs during germination (Fig. 2) must have a biological meaning other than just being a requirement for ARP Rb assembly.

On the other hand, the in vitro translation rates observed when phosphorylated versus nonphosphorylated Rbs were tested strongly suggest that the ARP's phosphorylation role is related to translational regulation (Fig. 5). In these experiments the Rb ARP phosphorylation status was the determinant for translation efficiency; faster in vitro translation rates were measured in the system containing phosphorylated ARP Rbs than in the nonphosphorylated ARP-Rb system (Fig. 5). These results were observed regardless of S6 RP phosphorylation inhibition (Fig. 5B).

Considering the overall data presented here, it might be concluded that neither ARP de novo synthesis nor Rb ARP assembly are determinant events for ARP phosphorylation and protein synthesis reinitiation during germination. On the other hand, ARP phosphorylation might have a relevant regulatory translation role in this specific developmental stage of maize axes.

ACKNOWLEDGMENTS

The authors thank Dr. J.P.G. Ballesta for the yeast ARP antibodies and for providing laboratory and technical facilities to perform IEF analysis of ARP Rbs.

Abbreviations:

ARP(s)

acidic ribosomal protein(s)

Rb(s)

ribosome(s)

Footnotes

1

This work was supported by the Dirección General de Asuntos del Personal Académico, Universidad Nacional Autonoma de Mexico (grant nos. IN200793 and IN217496).

LITERATURE  CITED

  1. Bailey-Jerres J, Vangala S, Szick K. Acidic ribosomal protein complex of the 60S ribosomal subunit of maize seedling roots. Plant Physiol. 1997;114:1293–1305. doi: 10.1104/pp.114.4.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballesta JPG, Remacha M. The large ribosomal stalk as a regulatory element of the eukaryotic translational machinery. Prog Nucleic Acid Res Mol Biol. 1996;55:157–193. doi: 10.1016/s0079-6603(08)60193-2. [DOI] [PubMed] [Google Scholar]
  3. Beltrame M, Bianchi ME. A gene family for acidic ribosomal proteins in Schizosaccharomyces pombe: two essential and two non essential genes. Mol Cell Biol. 1990;10:2341–2348. doi: 10.1128/mcb.10.5.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beltrán E, Ortiz-López A, Sánchez de Jiménez E. Synthesis of ribosomal proteins from stored mRNAs early in seed germination. Plant Mol Biol. 1995;28:327–336. doi: 10.1007/BF00020251. [DOI] [PubMed] [Google Scholar]
  5. Bermejo B, Ramacha M, Ortiz-Reyes B, Santos C, Ballesta JPG. Importance of the 5′ untranslated region of the mRNA for acidic ribosomal phosphoproteins on gene expression and protein accumulation. J Biol Chem. 1994;269:3968–3973. [PubMed] [Google Scholar]
  6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Goddemeier ML, Rensing SA, Felix G. Characterization of a maize ribosomal P2 protein cDNA and phylogenetic analysis of the P1/P2 family of ribosomal proteins. Plant Mol Biol. 1996;30:655–658. doi: 10.1007/BF00049340. [DOI] [PubMed] [Google Scholar]
  8. Hershey JBW. Protein phosphorylation controls translation rates. J Biol Chem. 1989;254:20823–20826. [PubMed] [Google Scholar]
  9. Jefferies HBJ, Reinhard C, Kozma SC, Thomas G. Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family. Proc Natl Acad Sci USA. 1994;91:4441–4445. doi: 10.1073/pnas.91.10.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Juan Vidales F, Sánchez-Madrid F, Ballesta JPG. The acidic proteins of eukaryotic ribosomes: a comparative study. Biochim Biophys Acta. 1981;656:28–35. doi: 10.1016/0005-2787(81)90022-8. [DOI] [PubMed] [Google Scholar]
  11. Juan-Vidales F, Saenz-Robles MT, Ballesta JPG. Acidic protein of the large ribosomal subunit in Saccharomyces cerevisiae. Biochemistry. 1984;23:390–396. doi: 10.1021/bi00297a032. [DOI] [PubMed] [Google Scholar]
  12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. MacConnell WP, Kaplan NO. The activity of the acidic phosphoproteins from 80S rat liver ribosome. J Biol Chem. 1982;257:5359–5366. [PubMed] [Google Scholar]
  14. Memelink J, Swords KMM, Staehelin LA, Hoge JHC. Southern, northern and western blot analysis. Plant Mol Biol Manual. 1994;FI:1–23. [Google Scholar]
  15. Mitsui K, Nakagawa T, Tsurugi K. On the size and the role of a free cytosolic pool of acidic ribosomal proteins in yeast Saccharomyces cerevisiae. J Biochem. 1988;104:908–911. doi: 10.1093/oxfordjournals.jbchem.a122581. [DOI] [PubMed] [Google Scholar]
  16. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. 1962;15:473–497. [Google Scholar]
  17. Naranda T, Ballesta JPG. Phosphorylation controls binding of acidic proteins to the ribosome. Proc Natl Acad Sci USA. 1991;88:10563–10567. doi: 10.1073/pnas.88.23.10563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pérez-Méndez A, Aguilar R, Briones E, Sánchez de Jiménez E. Characterization of ribosomal protein phosphorylation in maize axes during germination. Plant Sci. 1993;94:71–79. [Google Scholar]
  19. Petermann ML. The dissociation of rat liver ribosomes to active subunits by urea. In: Moldave K, Grossman L, editors. Methods in Enzymology, London: Academic Press; 1971. pp. 429–433. [DOI] [PubMed] [Google Scholar]
  20. Price DJ, Grove JR, Calvo V, Auruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992;257:973–977. doi: 10.1126/science.1380182. [DOI] [PubMed] [Google Scholar]
  21. Ramjoué HP, Gordon J. Evolutionary microdivergence of chicken and rat liver ribosomal proteins. J Biol Chem. 1977;252:9065–9070. [PubMed] [Google Scholar]
  22. Remacha M, Jiménez-Díaz A, Bermejo B, Rodriguez-Gabriel MA, Guarinos E, Ballesta JPG. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae. Mol Cell Biol. 1995a;15:4754–4762. doi: 10.1128/mcb.15.9.4754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Remacha M, Jiménez-Díaz A, Santos C, Briones E, Zambrano R, Rodríguez Gabriel MA, Guarinos E, Ballesta JPG. Proteins P1, P2 and P0 components of the eukaryotic ribosome stalk: new structural and funtional aspects. Biochem Cell Biol. 1995b;73:959–968. doi: 10.1139/o95-103. [DOI] [PubMed] [Google Scholar]
  24. Remacha M, Santos C, Ballesta JPG. Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae. Mol Cell Biol. 1990;10:2182–2190. doi: 10.1128/mcb.10.5.2182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Saenz-Robles MT, Remacha M, Vilella MD, Zinker S, Ballesta JPG. The acidic ribosomal proteins regulators of the eukaryotic ribosomal activity. Biochim Biophys Acta. 1990;1050:51–55. doi: 10.1016/0167-4781(90)90140-w. [DOI] [PubMed] [Google Scholar]
  26. Sánchez de Jiménez E, Aguilar R, Dinkova T. S6 ribosomal protein phosphorylation and translation of stored mRNA in maize. Biochimie. 1997;79:187–194. doi: 10.1016/s0300-9084(97)83505-5. [DOI] [PubMed] [Google Scholar]
  27. Sánchez-Madrid F, Juan Vidales F, Ballesta JPG. Effect of phosphorylation on affinity of acidic ribosomal proteins from Saccharomyces cerevisiae for the ribosome. Eur J Biochem. 1981;114:609–613. doi: 10.1111/j.1432-1033.1981.tb05187.x. [DOI] [PubMed] [Google Scholar]
  28. Sánchez-Madrid F, Reyes R, Conde P, Ballesta JPG. Acidic ribosomal proteins from eukaryotic cells: effect on ribosomal function. Eur J Biochem. 1979;98:409–416. doi: 10.1111/j.1432-1033.1979.tb13200.x. [DOI] [PubMed] [Google Scholar]
  29. Sepúlveda G, Aguilar R, Sánchez de Jiménez E. Purification and partial characterization of an acidic ribosomal protein kinase from maize. Physiol Plant. 1995;94:715–721. [Google Scholar]
  30. Sharf K, Nover L. Heat shock-induced alterations of ribosomal protein phosphorylation in plant cell cultures. Cell. 1982;30:427–437. doi: 10.1016/0092-8674(82)90240-9. [DOI] [PubMed] [Google Scholar]
  31. Shimmin LC, Ramirez G, Matheson AT, Dennis PP. Sequence alignment and evolutionary comparison of the L10 equivalent and L12 equivalent ribosomal proteins from archaebacterial, eubacterial and eukaryotes. J Mol Evol. 1989;29:448–462. doi: 10.1007/BF02602915. [DOI] [PubMed] [Google Scholar]
  32. Strycharz WA, Nomura M, Lake JA. Ribosomal proteins L7/L12 localized at the single region of the large subunit by immune electronmicroscopy. J Mol Biol. 1978;126:123–140. doi: 10.1016/0022-2836(78)90355-8. [DOI] [PubMed] [Google Scholar]
  33. Terada N. Patel HR, Takase K, Kohno K, Nair AC, Gelfand EW (1994) Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci USA 91: 11477–11481 [DOI] [PMC free article] [PubMed]
  34. Uchiumi T, Traut RR, Kominami R. Monoclonal antibodies against acidic phosphoproteins P0, P1 and P2 of eukaryotic ribosomes as functional probes. J Biol Chem. 1990;265:89–95. [PubMed] [Google Scholar]
  35. Vázquez MP, Schijman AG, Pametra A, Levin MJ. Nucleotide sequence of a cDNA encoding another Trypanosoma cruzi acidic ribosomal P2 type protein (Tc P2b) Nucleic Acids Res. 1992;20:2893. doi: 10.1093/nar/20.11.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wool IG, Chan YL, Glück A, Suzuki K. The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins. Biochimie. 1991;73:861–870. doi: 10.1016/0300-9084(91)90127-m. [DOI] [PubMed] [Google Scholar]
  37. Zinker S, Warner J. The ribosomal proteins of Saccharomyces cerevisiae: phosphorylated and exchangeable proteins. J Biol Chem. 1976;251:1799–1807. [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES