From the Department of Botany and Plant Sciences, University of California, Riverside, California 92521-0124
Received for publication, December 6, 2000
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ABSTRACT |
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Maize (Zea mays L.) possesses
four distinct ~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.
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 (~35 kDa), homologous to prokaryotic L10,
interacts with 28 S 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 (2-5). P1 and P2 are structurally similar; each protein has
three domains that include an 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). Saenz-Robles
et al. (12) 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 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 (P1 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 that 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 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
(Z. 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, and 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 4-5 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
N2 and stored at RNA Isolation and Northern Hybridization--
Total RNA was
extracted following a CsCl gradient method (29), and RNA blots (20 µg
of RNA/sample) were prepared (30). Hybridization was with
[ Isolation of Ribosomes and Soluble Proteins and Purification of
Acidic Ribosomal Proteins--
1-10 g of tissue was ground to a fine
powder under liquid N2 with a mortar and pestle and
hydrated in 2-20 ml of extraction buffer A (0.2 M Tris, pH
7.5, 0.2 M KCl, 0.025 M EGTA, 0.036 M MgCl2, 0.001 M
Na2MoO4, 0.001 M
dithiothreitol, 50 µg/ml cycloheximide, 50 µg/ml
chloramphenicol) containing a detergent mix (1% Triton X-100 (v/v),
1% Brij 35 (w/v), 1% Tween 40 (v/v), 1% IGEPAL CA-630 (tert-octylphenoxy poly(oxyethylene) ethanol) (v/v)). The homogenate was centrifuged at 4 °C for 20 min at 7,740 × g,
and the resulting supernatant (S-8 extract) was filtered
(Miracloth; Calbiochem). Ribosomes were isolated by centrifugation of
the S-8 extract through a 1.3 M sucrose cushion (0.4 M Tris, pH 7.5, 0.2 M KCl, 0.005 M
EGTA, 0.036 M MgCl2, 0.001 M
Na2MoO4, 0.001 M dithiothreitol, 50 µg/ml cycloheximide, 50 µg/ml chloramphenicol) at 4 °C for 18-20 h at 135,000 × g. Ribosome pellets were
resuspended in 0.02 M Tris, pH 7.5, 0.1 M KCl,
0.005 M MgCl2, 0.001 M
Na2MoO4, and 0.001 M
dithiothreitol. To analyze zeins, S-8 extracts were prepared from endosperm tissue in the same manner, except that cycloheximide and
chloramphenicol were omitted from the extraction buffer. Protein concentration was determined by the Bradford method using a protein determination reagent (United States Biochemical Corp.).
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). 1-10 g of tissue was ground to a fine powder under
liquid N2 with a mortar and pestle and hydrated in 2-20 ml
of extraction buffer B (0.2 M Tris, pH 7.5, 0.06 M KCl, 0.05 M MgCl2, 0.005 M dithiothreitol, 50 µg/ml cycloheximide). The homogenate
was filtered through Miracloth and centrifuged at 4 °C for 10 min at
37,000 × g. Detergents (as described above) were added
to the supernatant, which contained soluble ribosomes. The pellet,
which contained membrane-bound ribosomes, was resuspended in extraction
buffer containing detergents. Ribosomes were isolated from each
fraction by centrifugation through a 1.3 M sucrose cushion in extraction buffer B at 4 °C for 18-20 h at 135,000 × g and resuspended in extraction buffer B minus cycloheximide.
SDS-Polyacrylamide Gel Electrophoresis of Proteins and Immunoblot
Analysis--
Ribosomes (2.5 µg) and S-8 extract protein (75 µg) were diluted with SDS-sample buffer to a final concentration of
0.005 M Tris, pH 6.8, 5% glycerol (v/v), 2% SDS (w/v),
0.5%
Membranes were blocked for 1 h in PBST (PBS (31), 0.1% Tween 20 (v/v)) that contained 5% nonfat dry milk (w/v) and incubated with
rabbit antiserum against maize S6 (1:5,000 dilution) (kindly provided
by A. Williams), rabbit antisera against maize 12-kDa P-protein
peptides (P1 (1:250 dilution), P2a (1:250 dilution), P2b (1:250
dilution), P3 (1:1,000 dilution)), or rabbit antiserum against maize
Production of Polyclonal Antibodies--
Peptides specific to
maize P-proteins (44ALFAKLLEKRNVED57 for
P1, 40ELLLSQLSGKD50 for P2a,
39LEFLLTELKDKDI51 for P2b, and
9RNNGGEWTAKQHSGEI24 for P3) were synthesized,
conjugated with a carrier protein (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 (Fig.
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 18 S 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 NH4Cl and 50%
ethanol) under conditions that release the 12-kDa P-proteins (26), and
immunoblot analyses were performed. Preimmune 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 (Fig.
2A, lane 1 in each
panel).
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 (Fig. 2A,
P1 panel, lane 1). A low level of P1 remained
in the salt-washed ribosomes (lane 2). Following release
from ribosomes with 0.8 M NH4Cl, P1 was
detected primarily as 15 and 14.5 kDa (lane 3). The
50-kDa protein detected with the P1 antiserum was not released from
ribosomes (lane 3). This protein was identified as r-protein L4 by matrix-assisted laser absorption ionization-time of flight mass
spectrometry (data not shown), indicating that the detection of the
50-kDa protein is most likely caused by nonspecific binding of the P1 antiserum.
Maize P2a is encoded by a gene family of approximately four members,
has a predicted molecular mass of 11.5 kDa, and five or six putative
phosphorylation sites (26, 27). The P2a antiserum detected a group of
~12-kDa polypeptides in coleoptile ribosomes (Fig. 2A,
P2a panel, lane 1). The detection of multiple
12-kDa forms of P2a is most likely caused by 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 (Fig. 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 (24-26
kDa) and additional multimeric complexes (42-48 kDa, 60 kDa) in
coleoptile ribosomes.
Maize P2b is encoded by a single gene, has a predicted molecular
mass of 11.8 kDa, and three putative phosphorylation sites (26,
27). P2b was detected as a 14-kDa polypeptide in coleoptile ribosomes
(Fig. 2A, P2b panel, lane 1) and was
efficiently released from ribosomes by 0.8 M
NH4Cl (lane 2). Similar to P1, P2b was detected
after elution as 14- and 14.5-kDa forms (lane 3).
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 (Fig.
2A, P3 panel, lane 1). When P3 was released with 0.8 M NH4Cl two forms of 15.5 and
15 kDa were detected (lane 3). Much of the P3 protein
remained in the salt-washed ribosomes (lane 2), indicating
that the noncovalent interactions between P3 and the ribosome are
stronger than that observed for P1, P2a, and P2b.
Electrophoretic variants of the different P-proteins and P2a complexes
were observed between different tissues (Fig.
3, discussed below) and following release
from coleoptile ribosomes with 0.8 M NH4Cl
(Fig. 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 Fig. 2B. To confirm loading of similar quantities of
r-proteins in each sample, immunoblots were coincubated 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 (Fig. 2B). A
panel for P2b is not shown because this form was not detected in root
tip ribosomes (see Fig. 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 (Fig. 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
ribosome-associated kinase to phosphorylate coleoptile P-proteins was
evidenced by the change in electrophoretic mobility following
resuspension in a buffer lacking sodium molybdate (Fig. 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 ribosome-associated kinases.
12-kDa P-protein Phosphorylation Is Reduced in Response to
Anoxia--
Our previous investigations indicated that the
phosphorylation status of the 12-kDa P-proteins is altered in response
to anoxia (26). The immunoblot shown in Fig. 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 demonstrates
unambiguously 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 Fig. 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.
P1 was detected in ribosomes of all of the samples but in different
amounts and in several electrophoretic variants (Fig. 3, panel
P1). P1 was less abundant in leaf, silk, and ear ribosomes, compared with root, coleoptile, and kernel tissues. Variations in P1
included the presence of a single form (15 kDa) in leaf, silk, ear,
embryo, and endosperm, two forms in root tip (15 and 14.5 kDa) and
coleoptile (14.5 and 12 kDa), and three forms (15, 13, and 12 kDa) in
aleurone ribosomes. As demonstrated in Fig. 2B, the 15- and
14.5-kDa forms differ in phosphorylation status. The 13- and 12-kDa
forms detected in coleoptiles and aleurone were present at varying
levels between preparations and are most likely degradation products.
The P2a antiserum detected polypeptides that varied considerably in
abundance and electrophoretic mobility between the different organ and
tissue samples (Fig. 3, panel P2a). The 12.5-kDa forms of
P2a were abundant in root tip, embryo, and endosperm ribosomes, whereas
a 12-kDa form was detected in coleoptile ribosomes. Consistent with
mRNA accumulation data, lower levels of P2a were detected in leaf,
silk, ear, and aleurone ribosomes. Levels of dimeric (24-26 kDa) and
multimeric P2a (48 and 60 kDa) were proportional to levels of the 12- and 12.5-kDa P2a in all samples except ribosomes of embryo and
endosperm. P2a 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 (Fig. 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 (Fig. 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 because of reduced
phosphorylation (Fig. 3, P3 panel). A small amount of
dephosphorylated P3 was detected consistently in root tip ribosomes.
The levels of P1 and P3 in leaf, silk, and ear ribosomes (Fig. 3) are
higher than predicted from the transcript accumulation data (Fig.
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. Fig.
4,A-C, compares the P-protein
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 altered dramatically (Fig. 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) (Fig. 4B). An increase in the electrophoretic mobility of P2a that was consistent with dephosphorylation was observed
at 40 DPP. The level of P2b was modulated less dramatically 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 12-15 DPP, at which time
grain filling begins and proceeds until ~40-50 DPP, when a spatial
progression in programmed cell death occurs (34). We observed that
levels of P1, P2a, and P3 were not regulated markedly during endosperm
development (Fig. 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 because of a reduction in
ribosome levels at the end of endosperm maturation (data not shown).
Distinctions in Ribosomes Associated with Storage Protein Bodies of
Endosperm--
Large quantities of storage proteins are synthesized
during endosperm maturation. Prolamines ( 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 caused by 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 versus 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 nonspecific
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 60 S (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 P1
Yeast strains in which the phosphorylated C-terminal serine of P1 Ribosome Heterogeneity Is Caused Mainly by 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 compared with 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
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 eluted less
efficiently. 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 the 12-kDa P-proteins of
maize ribosomes and that these distinctions are affected by cell
identity, growth conditions, and stage of development. These results
raise several intriguing questions: 1) Do maize ribosomes possess each of the three types of 12-kDa P-proteins (P1, P2, and P3) at similar or
distinct levels? 2) Is there a heterogeneous population of ribosomes
within a plant cell (i.e. ribosomes with different levels of
the 12-kDa P-proteins)? 3) Are differences in abundance and phosphorylation of the P-proteins involved in translational regulation?
Ribosome heterogeneity in yeast was considered by Ballesta et
al. (46) when they examined the P-protein complex by affinity purification of ribosomes with His-tagged P2
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 (Fig. 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 (types
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. Because 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 caused by 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
and P1
) and P2 (P2
and P2
) (21). Mutant yeast strains in which
one to four of the 12-kDa P-protein genes were disrupted remained viable but had decreased rates of cell growth (20, 22, 23). 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 profiles of proteins
synthesized from the same poly(A) mRNA sample with ribosomes from
this strain and a wild-type strain were distinct, suggesting that the
presence of P1 and P2 influences the efficiency of translation of
individual mRNAs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
80 °C.
-32P]dATP-labeled cDNAs (GenBank U62752, maize
P1; T18290, maize P2a; U62753, maize P2b; U62751, maize P3; tomato 18 S
rRNA (gift of Dr. D. Bird)) separately, overnight at 42 °C in 6 × SSC, 5 × Denhardt's, 0.5% SDS (w/v), 100 µg/ml denatured
calf thymus DNA, and 50% formamide (v/v) (31), washed twice in 2 × SSC and 0.1% SDS (w/v), once in 0.2 × SSC, and 0.1% SDS
(w/v) for 20 min each at 65 °C, and exposed to autoradiographic film (Hyperfilm; Amersham Pharmacia Biotech) overnight at
80 °C
with an intensifying screen. Prior to reuse, membranes were washed in
0.05 × SSC, 0.01 M EDTA, pH 8.0, and 0.1% SDS (w/v)
for 20 min at 100 °C.
-mercaptoethanol, and 0.125% bromphenol blue (w/v). Samples
were heated at 100 °C for 5 min, and insoluble material was removed
by centrifugation. Ribosomal proteins were fractionated in resolving
gels (15% acrylamide (w/v), 0.5% (w/v)
N,N'-methylene-bisacrylamide, 0.375 M
Tris, pH 8.8, 0.1% SDS (w/v), 0.5% ammonium persulfate (w/v), and
0.5% TEMED). To examine zein levels, the S-8 extract was
fractionated in 12% acrylamide (w/v), 0.5% (w/v)
N,N'-methylene-bisacrylamide gels. Proteins were
electrophoresed in SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS (w/v) and electrophoretically transferred to nitrocellulose membranes (0.22 µm; NitroBind; Micron Separations Inc.) in 0.025 M Tris, 0.250 M
glycine, 20% methanol (v/v), and 0.01% SDS (w/v).
-zein (1:10,000 dilution) (kindly provided by A. Esen, North
Carolina State University) in PBST, 1% nonfat dry milk (w/v) for
1 h. Membranes were washed three times for 5 min in PBST and
incubated for 1.5 h in PBST, 1% nonfat dry milk (w/v) with goat
anti-rabbit IgG horseradish peroxidase conjugate (1:15,000 dilution;
Bio-Rad) or 35S-labeled donkey anti-rabbit IgG (2 × 105 cpm/ml; Amersham Pharmacia Biotech). Antibody-antigen
interaction was detected by chemilumenscence using the ECL reagent
(Amersham Pharmacia Biotech). Membranes that were incubated with
35S-labeled anti-rabbit IgG were exposed to a
PhosphorImager screen (Molecular Dynamics) and were quantified using
QuantityOne software (Bio-Rad).
RESULTS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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View larger version (74K):
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Fig. 1.
Developmental regulation of 12-kDa P-protein
mRNA accumulation. Total RNA was isolated from various
tissues, separated (20 µg/lane), blotted, and sequentially
hybridized at high stringency with 32P-labeled cDNAs
encoding P1, P2a, P2b, P3, and 18 S rRNA.
View larger version (55K):
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Fig. 2.
Detection of maize P1, P2a, P2b, and P3 with
specific antisera. Panel A, ribosomes isolated from
coleoptiles (lane 1), acidic proteins released from
coleoptiles ribosomes with 0.8 M NH4Cl
(lane 3), and corresponding salt-washed ribosomes
(lane 2) were resolved by SDS-PAGE, transferred to
nitrocellulose, and were incubated with P1, P2a, P2b, or P3 antisera.
The black arrow indicates the position of the monomeric
proteins. White, gray, and stippled
arrows indicate the P2a dimeric and multimeric complexes,
respectively. Panel B, ribosomes isolated from root tips in
the presence (+) or absence ( ) of sodium molybdate were resolved by
SDS-PAGE, transferred to nitrocellulose, and incubated simultaneously
with antisera against S6 (as indicated) and P1, P2a, or P3.
White, gray, and stippled arrows
indicate the P2a dimeric and multimeric complexes, respectively.
Panel C, ribosomes isolated from aerobic, 6-h, 12-h, or 24-h
oxygen-deprived root tips in the presence of sodium molybdate were
resolved by SDS-PAGE, transferred to nitrocellulose, and incubated
simultaneously with antisera against S6 (as indicated) and P1, P2a, or
P3. The higher molecular mass forms of P2a were detected in all samples
after longer exposure (data not shown)
View larger version (63K):
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Fig. 3.
Detection of P1, P2a, P2b, P3, and S6 in
ribosomes from various tissues of maize. Ribosomes isolated from
various developmental tissues and organs (see "Experimental
Procedures") were resolved by SDS-PAGE, transferred to
nitrocellulose, and incubated simultaneously with antisera against S6
(indicated by black arrow) and P1, P2a, P2b, or P3.
White, gray, and stippled arrows
indicate the P2a dimeric and multimeric complexes, respectively. The
apparent molecular mass of each protein (kDa) is indicated on the
right as determined by the migration of molecular mass
markers.
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Fig. 4.
Detection of P1, P2a, P2b, P3, and S6 in
ribosomes during maize kernel maturation. Ribosomes isolated from
(panel A) 15, 20, 25, 30, and 40 DPP and dry embryos;
(panel B) 10-, 15-, 20-, 25-, 30-, and 40-DPP aleurone; and
(panel C) 10-, 15-, 20-, 25-, 30-, and 40-DPP endosperm were
resolved by SDS-PAGE, transferred to nitrocellulose, and incubated
simultaneously with antisera against S6 and P1, P2a, P2b, or P3. The
apparent molecular mass (kDa) of each protein is indicated on the
right.
,
,
,
-zeins) are
alcohol-soluble proteins that are synthesized on rough endoplasmic
reticulum and are assembled into protein bodies (PBs) in the lumen.
Zein accumulation begins at about 10 DPP but accelerates only after 25 DPP (Fig. 5; 35), despite a peak in zein
mRNA levels at 15-DPP (35, 36), indicative of post-transcriptional
regulation of expression. We observed that ribosomes isolated from 25-, 30-, and 40-DPP endosperm were contaminated with zeins. This was not
unexpected because prolamine PBs are found tightly associated with the
cytoskeleton and rough endoplasmic reticulum in maize and rice (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 recentrifuged 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
pelleted again. This pellet contained high levels of zeins and
r-proteins, confirming the purification of a fraction enriched in
PB-associated ribosomes (Fig. 6,
-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 (Fig. 6). In coleoptiles,
slightly reduced levels of these proteins were reproducibly detected in
membrane-associated ribosomes. This was especially evident for the P2a
monomer (Fig. 6) and dimer (data not shown). In the 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 with soluble ribosomes, and P2b was undetectable in
PB-associated ribosomes. 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.
View larger version (55K):
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Fig. 5.
Accumulation of
-zein seed storage proteins during endosperm
development. Panel A, soluble protein isolated from
10-, 15-, 20-, 25-, 30-, and 40-DPP endosperm was resolved by SDS-PAGE.
Proteins were visualized by Coomassie Blue staining. The positions of
molecular mass markers (kDa) are indicated on the left.
Panel B, soluble protein isolated from 10-, 15-, 20-, 25-, 30-, and 40-DPP endosperm was resolved by SDS-PAGE, transferred to
nitrocellulose, and incubated with maize
-zein antiserum
(black arrows). Panel C, relative abundance of
-zein in endosperm development. Soluble protein isolated from 10-, 15-, 20-, 25-, 30-, and 40-DPP endosperm was resolved by SDS-PAGE,
transferred to nitrocellulose, and detected with maize
-zein
antiserum. The signals of the 35S-labeled cross-reacting
anti-rabbit IgG secondary antibody were quantified with a
PhosphorImager.
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Fig. 6.
Detection of P1, P2a, P2b, P3, and S6 in
membrane-associated and soluble ribosomes. Soluble (S),
membrane-associated (M), and zein protein
body-associated (PB) ribosomes isolated from coleoptiles or
40-DPP endosperm were resolved by SDS-PAGE, transferred to
nitrocellulose, and incubated simultaneously with S6, P1, P2a, P2b, P3,
or zein antiserum. The apparent molecular mass of each protein (kDa) is
indicated on the right as determined by the migration of
molecular mass markers.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and P2
, 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. Because all of the maize
P-proteins were modified during anoxia in the same manner, we predict
that this modulation is caused by 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 the result of the inactivation
of a kinase and/or activation of a phosphatase that specifically
modifies P2a.
was mutated to an alanine were less sensitive to osmotic stress than
wild-type 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 because dephosphorylation of the C-terminal serine of rat P2 reduced
translational activity in vitro (17). Additional studies are
needed to determine whether 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.
-helices with
hydrophobic residues spaced at intervals that would promote coiled-coil
interactions (45). Similar to yeast, maize P2a is predicted to form an
-helix with a strong hydrophobic edge that may result in the
observed dimers and
multimers.2 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.
. They determined that
95% of the ribosomes contained all four of the 12-kDa P-proteins (P1
, P1
, P2
, and P2
) and concluded that the yeast ribosomes are homogeneous with respect to P-protein composition. By
contrast, our results indicated that P1 and P3 levels were fairly
constant and coordinate, but levels of P2a and P2b were dramatically
variable in maize ribosomes even when levels of both P2a and P2b were
considered. Phylogenetic studies indicate that P2a and P2b are present
in maize and rice, indicating that these two forms arose from a gene duplication event prior to the divergence of a common ancestor and that
two classes of P2 are present in Arabidopsis.2
The significant heterogeneity in P2 levels and prevalence of multiple
P2 forms in plants may be indicative of a functional role of the
distinct P2 forms.
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Fig. 7.
Model of ribosome heterogeneity due to
variation in P2 protein composition of the acidic stalk. Type I
ribosomes possess a minimal P-protein complex with the stalk formed by
P0, P1, and P3 and exist during early aleurone development and in ears
prior to pollination. Variations in P2 assembly onto the minimal
P-protein complex result in ribosome types II-V. Type II ribosomal
stalks possess the minimal complex with the addition of the monomeric
form of P2a and predominate in kernel tissues. Type III ribosomal
stalks possess the minimal complex and dimers or multimers of P2a
(P2a*) and predominate in root and coleoptile. Type IV ribosomal stalks
possess the minimal complex and P2b and predominate in leaf and silk,
whereas type V ribosomal stalks possess the minimal complex and both
P2a and P2b and may exist in cells of coleoptile and other tissues that
contain P2a and P2b. Variations in stalk protein composition could
influence elongation rates (eEF2-dependent GTPase
activity). Regulated differences in phosphorylation of the 12-kDa
P-proteins provide additional ribosomal heterogeneity and potential
ramifications on translation.
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FOOTNOTES |
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* This research was supported by United States Department of Agriculture Grants 97-35100-4191 and 00-35301-9108 (to J. B.-S.), National Science Foundation Grant MCB 920192 (to J. B.-S.), and by funding from the Interdepartmental Graduate Program in Genetics (to K. S.-M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Botany and
Plant Sciences, 2130 Batchelor Hall, University of California, Riverside, CA 92521-0124. Tel.: 909-787-3738; Fax: 909-787-4437; E-mail: serres@mail.ucr.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M011002200
2 K. Szick-Miranda and J. Bailey-Serres, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: r-protein(s), ribosomal protein(s); DPP, days postpollination; TEMED, N,N,N',N'-tetramethylethylenediamine; PBS, phosphate-buffered saline; PB, protein body; PAGE, polyacrylamide gel electrophoresis.
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