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Plant Physiol. (1998) 116: 559-569
Evidence for a Cytoskeleton-Associated Binding Site Involved in
Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm
Tissue1
Douglas G. Muench2, 3,
Yujia Wu2,
Sean J. Coughlan, and
Thomas W. Okita*
Institute of Biological Chemistry, Washington State University,
Pullman, Washington 99164-6340 (D.G.M., Y.W., T.W.O.); and Pioneer Hi-Bred International, Johnston, Iowa 50131-1004 (S.J.C.)
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ABSTRACT |
Previous
studies have demonstrated that the mRNAs encoding the prolamine and
glutelin storage proteins are localized to morphologically distinct
membranes of the endoplasmic reticulum (ER) complex in developing rice
(Oryza sativa L.) endosperm cells. To gain insight about
this mRNA localization process, we investigated the association of
prolamine polysomes on the ER that delimit the prolamine protein bodies
(PBs). The bulk of the prolamine polysomes were resistant to extraction
by 1% Triton X-100 either alone or together with puromycin, which
suggests that these translation complexes are anchored to the PB
surface through a second binding site in addition to the
well-characterized ribosome-binding site of the ER-localized protein
translocation complex. Suppression of translation initiation shows that
these polysomes are bound through the mRNA, as shown by the
simultaneous increase in the amounts of ribosome-free prolamine mRNAs
and decrease in prolamine polysome content associated with the
membrane-stripped PB fraction. The prolamine polysome-binding activity
is likely to be associated with the cytoskeleton, based on the
association of actin and tubulin with the prolamine polysomes and PBs
after sucrose-density centrifugation.
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INTRODUCTION |
Unlike most cereals that accumulate predominantly one type of
storage protein, rice (Oryza sativa L.) seeds synthesize
both major classes of storage proteins, the prolamines and the
glutelins (globulins). Both storage protein types are initially
synthesized on ER membranes but are then stored as PBs in different
intracellular compartments (Muench and Okita, 1997 ). Prolamines are
translocated into the ER lumen, where they are sequestered, forming a
protein-inclusion granule delimited by the rough ER. Newly synthesized
glutelins are also transported into the ER lumen but are subsequently
sorted via the Golgi apparatus and deposited in the vacuole (Tanaka et al., 1980; Krishnan et al., 1986). Because of the cellular events leading to prolamine PB biogenesis, the rough ER can be viewed as being
composed of morphologically distinct subdomains, the PB-ER and the C-ER
(Li et al., 1993a). These subdomains are also functionally distinct in
that prolamine and glutelin mRNAs are differentially sorted and
translated on the PB-ER and C-ER, respectively, as shown by
biochemical, in vitro, and in situ hybridization experiments (Li et
al., 1993a). Therefore, rice is able to accommodate the synthesis and
packaging into separate PBs of both major classes of storage proteins
by translating these mRNAs on different ER subdomains. The mechanism
responsible for the localization of the storage protein mRNA on these
ER membrane types has yet to be identified.
mRNA localization has been extensively characterized in a wide variety
of animal cells, many of which are polarized cell types (St. Johnston,
1995). The cytoskeleton has a direct role in the transport and
anchoring of several of these mRNAs to specific intracellular
locations. Both the microtubule and the microfilament networks are
known to function together or independently in the localization of
these mRNAs (Yisraeli et al., 1990; Sundell and Singer, 1991; St.
Johnston, 1995). Actin mRNAs in the brown algae of the genus
Fucus are localized to the cell plate in the developing embryo (Bouget et al., 1996). This RNA localization, however, is
insensitive to cytoskeleton inhibitors and therefore may depend on some
other mechanism for localization.
In addition to mRNA localization, the cytoskeleton has also been
implicated in translation. Most of the polysomes in the cytosol are
thought to be associated with the microfilament component of the
detergent-resistant cytoskeleton scaffolding network (Pachter, 1992;
Hesketh, 1994). Much of this evidence has come from electron microscopy, in situ hybridization, and biochemical fractionation studies of detergent-treated cell extracts and cells treated with cytoskeleton-destabilizing agents such as cytochalasin. These results
suggest that many polysomes that were once considered "free" in the
cytosol are in fact associated with the cytoskeleton, and adds to the
view that the cytosol is divided into metabolically distinct
compartments formed by the orderly association of metabolites and
macromolecules (Pachter, 1992). Although much of this evidence is from
animal systems, recent evidence indicates that polysomes in plant cells
are also associated with the cytoskeleton. Biochemical evidence for
cytoskeleton-bound polysomes has come from studying pea stems and roots
and corn endosperm (Davies and Abe, 1989; Davies et al., 1993; Ito et
al., 1994). In addition to cytoskeleton-bound polysomes in plants,
there is also evidence for the association of polysomes to both the
cytoskeleton and the membranes (Davies et al., 1993; Stankovic et al.,
1993; Ito et al., 1994). Isolated maize PBs are enmeshed in F-actin,
and nonionic detergent treatment removes all of the fluorescently
labeled ER membrane and all of the phospholipids but retains almost all
of the polysomes and the cytoskeleton components (Davies et al., 1993;
Stankovic et al., 1993). Treatment with the ionic detergent
deoxycholate solubilizes the cytoskeleton and releases the polysomes.
These polysome types, termed cytomatrix-bound polysomes (Ito et al.,
1994), are also found in animal cells (Zambetti et al., 1990b).
Although it is clear that many polysomes are bound to the cytoskeleton,
the nature of this interaction is only poorly understood. In plant
cells there is some evidence for the role of the ribosome or perhaps
the nascent polypeptide chain in anchoring the polysome to the
cytoskeleton (You et al., 1992; Davies et al., 1993; Stankovic et al.,
1993). Treatment of animal cells with inhibitors of translation such as
fluoride results in an increase in ribosome-free mRNAs, but fails to
release these mRNAs from the cell matrix (Howe and Hershey, 1984; Bag
and Pramanik, 1987). This suggests that some polysomes are anchored not
through the ribosome but via the mRNA. In addition, many membrane-bound
polysomes are not released by treatment with cytochalasin or puromycin
alone, but when the inhibitors are used simultaneously, these
membrane-bound polysomes are released (Zambetti et al., 1990b). This
observation suggests that membrane-bound polysomes are anchored at two
sites, one associated with the cytoskeleton, and the other with the
well-characterized polypeptide translocation complex receptor that
binds the ribosome (Rapoport, 1992).
In an effort to understand the mechanism responsible for prolamine and
glutelin mRNA localization in rice endosperm cells, we used a
biochemical approach to study the interaction among the prolamine
polysomes, the PB-ER, and the cytoskeleton. Here we present evidence
for the existence of a second prolamine polysome-binding activity in
addition to the ribosome receptor of the polypeptide translocation
complex of the ER. We also show that this second prolamine
polysome-binding activity is likely to be associated with the
cytoskeleton and that it interacts not with the nascent polypeptide
chain but most likely with the mRNA itself.
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MATERIALS AND METHODS |
Buffers and Seed Extract Preparation
Seeds from rice (Oryza sativa L. var M 201) were
harvested 15 d after flowering (mid-developing), frozen in liquid
nitrogen, and stored at  80°C. For experimental analysis, 1 g
of seed was typically ground with a mortar and pestle in liquid
nitrogen and then in 3 mL of CSB (5 mm Hepes adjusted to pH
7.5 with 3.2 mm KOH, 10 mm MgOAc, 2 mm EGTA, 1 mm PMSF, 1 mm DTT, 1 unit mL 1 RNase inhibitor [Inhibit-Ace, 5 Prime 3 Prime, Inc., Boulder, CO], and 200 mm Suc
[modified from Abe and Davies, 1991]). Other buffers used were CSB
containing either 1% Triton X-100 or 1% PTE, or buffer U consisting
of 200 mm Tris-HCl, pH 8.5, 50 mm KOAc, 25 mm MgOAc, 2 mm EGTA, 100 µg
mL1 heparin, 2% PTE, and 1% sodium
deoxycholate (Davies et al., 1993). The crude seed extract was then
filtered through one layer of Miracloth (Calbiochem). All steps were
carried out on ice or at 4°C. A PB fraction was obtained by first
removing most of the large starch grains by centrifuging the extract at
100g for 5 s, and then centrifuging the resulting
supernatant at 500g for 10 min to obtain the PB fraction.
The PB fraction was then analyzed directly or after pelleting and
resuspension in other buffers.
Suc-Density Centrifugation
Aliquots of crude extracts or PB fractions were analyzed by
Suc-density centrifugation using either 15 to 60% or 20 to 80% Suc
gradients containing 5 mm Hepes adjusted to pH 7.5 with 3.2 mm KOH, and 10 mm MgOAc. The 15 to 60%
gradients were generated as described previously (Davies and Abe, 1995)
and were centrifuged at 250,000g in a SW-50 rotor (Beckman)
for 60 min. The 20 to 80% gradients were generated by overlayering
80:60:40:20% Suc solutions in a 1:2:2:1 volume ratio, respectively,
and allowing them to equilibrate into a continual gradient overnight at
4°C. The 20 to 80% gradients were centrifuged at either
300,000g in a SW-55 rotor (Beckman) for 60 min or
300,000g in a SW-41 rotor (Beckman) for 150 min. After
centrifugation an absorbance profile at 254 nm was obtained and
fractions were collected using a UV5 monitor and a gradient
fractionator (model 185, Isco, Lincoln, NE).
Membrane Solubilization and Analysis
The solubilization of membranes in PB fractions or Suc-density
gradient fractions was performed by incubating the samples in CSB
containing 1% Triton X-100 or 1% PTE, or in buffer U for 10 min on
ice. The efficiency of membrane solubilization was determined by
analyzing the FA composition in untreated and detergent-treated PB
fractions using GC, or by analyzing the release of the ER membrane protein calnexin (see "Protein and RNA Blotting"). The GC analysis was similar to that described previously (Miquel and Browse, 1992). A
PB fraction was isolated as described above, and resuspended in CSB
containing Triton X-100. The detergent-treated fraction was then
centrifuged to obtain a PB pellet and supernatant. The pellet was
resuspended in an equal volume of chloroform:methanol:formic acid
solution (10:10:1), whereas 1.5 volumes of chloroform:methanol:formic acid solution (10:10:1) was added to the supernatant followed by the
addition of 1.5 volumes of methanol. After incubation at 20°C
overnight, the samples were then centrifuged at high speed in a
clinical centrifuge at 4°C and the supernatant resuspended in
one-fourth volume of Hajras solution (1 m KCl, 0.2 m H3PO4). After
shaking briefly, the sample was centrifuged and the lower organic phase
isolated, dried under nitrogen, and resuspended in 100 to 500 µL of
chloroform. To an aliquot of the chloroform sample was added 10 µL of
a 17:0 FA standard (0.25 mg mL1) as an internal
control. The sample was dried and resuspended in 1 mL of sulfuric acid
solution and incubated at 80°C. Water (1.5 mL) and hexane (250 µL)
were added and the sample was vigorously agitated and then centrifuged.
Approximately 3 µL of the upper hexane phase was analyzed for FA
composition on a gas chromatograph (model 5890A, Hewlett-Packard). The
GC profile data were normalized to the internal 17:0 FA standard.
Inhibitor Treatment
Treatment of rice seeds with 100 µm cytochalasin B,
100 µm cytochalasin D, 100 µm nocodazole,
or 25 mm NaF was performed using either intact rice
panicles bearing mid-developing rice seeds or half-sectioned seeds. For
panicle treatment, panicles were cut under water and transferred to a
15-mL test tube containing Murashige and Skoog medium supplemented with
2% Suc and a mixture of 21 amino acids (Donovan and Lee, 1977).
Inhibitors were added to the medium and the tubes placed in a growth
chamber under 11 h of light and 13 h of dark (30 h for the
cytochalasin B and D and nocodazole treatments and 6 h for the NaF
treatment). For half-seed treatments, seeds were sectioned
longitudinally with a razor blade and placed in the same Murashige and
Skoog medium with the appropriate drug and incubated in the dark at
26°C (6 h for the cytochalasin B and D treatment and 4 h for the
NaF treatment). Seeds from these treatments were then frozen in liquid
nitrogen and stored at 80°C and later processed as described above.
Protein and RNA Blotting
Protein and RNA were extracted from the same Suc- gradient
fraction after the addition of one volume of phenol. For RNA
extraction, the aqueous phase was extracted with
phenol:chloroform:isoamyl alcohol (24:24:1, v/v) and the RNA in the
aqueous phase was precipitated by the addition of 0.1 volume of sodium
acetate and 2 volumes of 95% ethanol. The sample was placed at
20°C for at least 1 h, and the RNA was then pelleted in a
microcentrifuge, washed with 70% ethanol, and resuspended in TE buffer
(10 mm Tris-HCl, pH 7.5, and 1 mm
Na2EDTA). For protein extraction, 3 to 5 volumes of
methanol containing 0.77% ammonium acetate was added to the organic
phase and the protein samples were incubated overnight at 20°C. The
protein was pelleted in a microcentrifuge, washed in acetone, and
resuspended in protein gel-loading buffer (Sambrook et al., 1989).
RNA samples were dot blotted to nitrocellulose along with known amounts
of prolamine cDNA as standards and hybridized with radiolabeled
prolamine or glutelin cDNA overnight at 65°C and washed under
stringent conditions. RNA levels were quantified either by
densitometric measurement of the radiographic signals or by phosphor
imager (Bio-Rad) analysis. Direct comparison of these two methods
showed that the results agreed within 5%. The protein samples were
boiled for 5 min and then run on 5 to 15% SDS-PAGE gradient gels with
a 4% stacking gel and transferred to nitrocellulose. The blots were
probed with rice prolamine antisera, soybean calnexin antisera, or
commercially available mouse anti- -tubulin monoclonal antibody
(T-9026, Sigma) and mouse anti-actin monoclonal antibody (N350,
Amersham; 691001, ICN). Secondary antibodies were conjugated to
horseradish peroxidase and were visualized by chemiluminescent detection (Super Signal chemiluminescence kit, Pierce).
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RESULTS |
Detergent and Salt Treatment of PB Fractions Indicates the Presence
of Non-ER/Polysome-Binding Activity
Differential centrifugation of endosperm tissue extracted in CSB
was used to isolate an enriched PB fraction. After filtration of the
crude extract, most of the starch grains were removed by centrifugation
at 100g for 5 min and an enriched PB fraction was obtained
by centrifugation at 500g for 10 min. This fraction, although enriched with PBs, also contained a significant amount of C-ER
membranes, as visualized by electron microscopy (Li et al., 1993a). The
membranes in this PB fraction were solubilized by the addition of 1%
Triton X-100 in CSB and the extract was then recentrifuged. The
supernatant was removed and buffer U was added to the pellet to release
the remaining bound polysomes. Polysomes were isolated from the pellet
and the supernatant of the detergent extract by centrifugation at
200,000g for 3 h through a 60% Suc pad. The polysome
pellets were solubilized in buffer U and quantified by
A260. Only 22% of the total polysomes were released from the PB fraction by detergent treatment (Table
I).
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Table I.
Percentage release of polysomes and FAs from
the detergent-washed PB fraction
A PB fraction was isolated in CSB as described in "Materials and
Methods," and then washed in CSB plus Triton X-100 and
recentrifuged at 500g for 10 min to yield a pellet and a
supernatant fraction.
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To determine the effectiveness of detergent treatment in solubilizing
the membrane from the PB pellet, FA analysis was performed using GC.
This analysis demonstrated that greater than 95% of each of the FAs
(16:0, 18:1, 18:2, and 18:3) were found in the supernatant fraction of
the detergent extract (Table I). These FAs constitute greater than 90%
of the total FAs in the PB fraction and, therefore, are the major FA
components of the membrane in this fraction. Protein gel-blot analysis
and immunodetection of the ER membrane-bound chaperone calnexin
demonstrated that calnexin is effectively released from the PB pellet
after 1% Triton X-100 treatment (Fig.
1). This treatment, therefore, would also
solubilize the ribosome receptor of the ER membrane translocation
complex (Rapoport, 1992), thus releasing the polysomes from their ER
membrane attachment site. Retention of the majority of polysomes in the detergent-washed pellet indicates that most of the polysomes in the PB
fraction are associated with the PB surface at a second site, in
addition to the ribosome-binding site on the ER membrane translocation
complex.
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| Figure 1.
Protein gel blot demonstrating the release of
calnexin from the detergent-washed PB fraction. A crude extract was
obtained by grinding 1 g of seeds in 3 mL of CSB (lane 1). The
extract was centrifuged at 500g, and the pellet was
resuspended in 3 mL of CSB plus 1% Triton X-100 (the detergent
extract, lane 2). The detergent extract was centrifuged to produce the
pellet (resuspended in 3 mL of CSB plus Triton X-100; lane 3) and the
supernatant (lane 4). Equal volumes of sample were loaded in each lane.
Protein gel blots were probed with calnexin antisera. The upper bands correspond to the ER membrane-bound chaperone calnexin, and the lower
bands correspond to the ER lumenal chaperone calreticulin, with which
the calnexin antisera cross-reacts.
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To determine if the anchoring of polysomes to the PB fraction is
mediated by the nascent polypeptide or the RNA component of the
polysomes, the PB fraction was washed twice with 1% Triton X-100 and
then incubated at 25°C for 15 min in CSB containing 1 mm
puromycin, a drug that mediates premature release of the polypeptide
chain from the ribosome. We have previously shown that 1 mm
puromycin effectively releases the nascent polypeptide chain from
polysomes in rice seed extracts under similar conditions (Li et al.,
1993b). Puromycin had no effect on the release of polysomes or mRNA
from detergent-treated PB fraction (Fig.
2A), indicating that the interaction
between the polysomes and the PBs is not mediated by the nascent
prolamine polypeptide. Polysomes associated with the membrane-stripped
PB fraction were, however, released by various solutions of relatively
high ionic strength, including 200 mm Tris-HCl, pH 7.5, 2%
ammonium sulfate, or 200 mm NaCl (Fig. 2A).
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| Figure 2.
Release of RNA and polysomes from the
detergent-treated PB fraction. A, The detergent-treated PB fraction was
incubated at 4°C for 15 min in the absence (control) or in the
presence of 200 mm NaCl, 2%
(NH4)2SO4, or 200 mm
Tris-HCl, pH 7.5, and then centrifuged at 500g for 10 min. The RNA in the pellet and supernatant was isolated by
phenol:chloroform extraction and ethanol precipitated, and then
quantified by A260. The 1 mm
puromycin-treated PBs were incubated in the absence of high salt
concentrations. Bars indicate se. B, Release of polysomes
from membrane-stripped PBs by NaCl. PBs were treated as above but in
the presence of varying concentrations of NaCl, and then the released
polysomes were fractionated through a 60% Suc pad, resuspended in
buffer U, and quantified by A260. The amount
of polysome release is estimated from A260
readings per gram of seed.
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To further characterize the effectiveness of salt on the release of the
polysomes from the PB fraction, we determined the concentration of NaCl
required to release the polysomes. An enriched PB fraction was treated
with varying concentrations of NaCl and then clarified by
centrifugation at 500g. At least 75 to 100 mm NaCl was required to release 50% of the total released polysomes (Fig.
2B). The 100 mm NaCl-released polysome fraction was
analyzed on a Suc-density gradient, and the absorbance profile revealed that the polysomes were intact, containing up to seven ribosomes per
transcript (data not shown). The above data suggest that the polysomes
are bound to the PB surface by a detergent-resistant, salt-sensitive
binding site either through the mRNA or ribosome, but not through the
nascent polypeptide chain.
Evidence for mRNA-Binding Activity
To elucidate whether the binding activity was interacting with the
mRNA or the ribosomes of the polysome complex, we determined whether
ribosome-free prolamine mRNAs could be associated with membrane-stripped PBs. As a strategy to increase the amount of cellular
mRNAs lacking ribosomes, developing seeds were first treated with 25 mm NaF before the mRNA composition of the membrane-stripped PBs was analyzed. NaF has been used in both animal (Hoerz and McCarty,
1971; Lenk et al., 1977) and plant (García-Hernández et
al., 1994) cells to dissociate polysome complexes by selectively inhibiting the initiation but not elongation step of protein synthesis. To test the efficiency of NaF on translation initiation, we monitored the incorporation of [3H]Leu into protein after
8, 12, 24, 36, and 48 h. In untreated seeds the incorporation rate
was linear for up to 36 h, whereas seeds treated with NaF showed
only a slight increase in incorporation after 8 h (data not
shown).
Membrane-stripped PB fractions isolated from control and 8-h
NaF-treated seeds were prepared and extracted with buffer U to release
the polysomes and ribosome-free mRNA from the PB fraction. The extract
was then subjected to high-speed centrifugation to pellet the PBs. The
clarified extract containing polysomes and ribosome-free mRNA was
loaded onto a 15 to 60% Suc gradient and then centrifuged at
250,000g for 60 min (Fig. 3A).
Fractions corresponding to soluble components, ribosomal subunits and
monosomes, and polysomes were isolated. Total RNA was isolated from
these fractions and the prolamine mRNA content was measured. A majority
(90% of the total) of the prolamine mRNA in the control profile was
present in the polysome (57%) and monosome (33%) fractions, with only a small amount (10%) present as ribosome-free species in the top soluble fraction (Fig. 3A). NaF treatment caused a decrease in the
amount of polysomes and an increase in the amount of monosomes (Fig.
3B).
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| Figure 3.
Suc-gradient profiles of PB fractions from control
seeds (A) or seeds treated with NaF (B). Seeds were treated with or
without 25 mm NaF as described in the text.
Membrane-stripped PBs were prepared and then washed in buffer U and
repelleted. Equal volumes of the supernatant from control and
NaF-treated seeds were layered onto 15 to 60% gradients. Bars
represent percent prolamine mRNA content in each of the fractions as
determined by dot-blot analysis. A se < 2.5% was
routinely obtained in quantifying mRNA levels. Fractions correspond to
soluble fraction (1) ribosomal subunit and monosome fraction (2), and
polysome fraction (3).
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This change in the polysome-monosome profile, however, had no effect on
the total prolamine mRNA content, which was nearly equivalent (within
4.5%) to the control profile. Consistent with the change in the
polysome-monosome profile, the distribution of prolamine mRNA was
altered, especially in the polysome fraction of the Suc gradient.
Prolamine mRNA content decreased approximately 25% in the polysome
fraction compared with the control. However, a corresponding increase
in prolamine mRNA content was evident in the soluble fraction, so that
all three fractions contained nearly the same levels of prolamine mRNA.
Hence, the prolamine mRNA percentages found in the NaF-treated samples
represent an authentic shift in prolamine mRNA from the polysomes to
ribosome-free mRNA rather than an artifact attributable to the
degradation of mRNA. Similar results were seen when mid-developing
seeds were sectioned longitudinally and then treated with 25 mm NaF in nutrient solution for 4 h. The increase in
prolamine mRNA in the soluble fraction of the NaF gradient indicates
that ribosome-free prolamine mRNA remains associated with
membrane-stripped PBs. These results suggest that ribosome-free
prolamine mRNA interacts with a putative RNA-binding activity near or
on the PB surface.
To further characterize the RNA-binding activity, membrane-stripped PBs
from NaF-treated seeds were extracted successively with 100, 200, and 300 mm NaCl and centrifuged to remove
the PBs. The supernatant of each of these treatments was then
fractionated through 15 to 60% Suc gradients and then assessed for
prolamine mRNA levels in the soluble fraction, ribosomal
subunit/monosome fraction, and polysome fraction (Fig.
4). Because of the much longer incubation
periods in this experiment, the polysomes were partially degraded,
which altered the distribution of ribosome-associated prolamine RNAs in
the Suc-density gradients. The bulk of the prolamine mRNA was
associated with the ribosomal subunit/monosome fraction in the 100 and
200 mm salt extracts instead of the polysome fraction, as
seen in Figure 3. Despite the apparent partial degradation of
polysomes, the patterns of released prolamine mRNAs in each of the
fractions from the three salt extractions were different (Fig. 4).
Prolamine mRNA that was associated with monosomes predominated in the
100 and 200 mm salt extracts, but were a smaller component of the 300 mm extract. Prolamine mRNA that was associated
with polysomes decreased in each of the successive salt-extraction steps, whereas ribosome-free prolamine mRNA was most efficiently released in the 300 mm salt fraction. Overall, these
results indicate the presence of a prolamine mRNA-binding activity that
requires relatively high ionic-strength levels to release the RNA from the PBs.
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| Figure 4.
Salt release of prolamine mRNA from NaF-treated
seeds. The membrane-stripped PB fraction from NaF-treated seeds was
extracted successively with 100, 200, and finally 300 mm
NaCl. The total supernatant from each extract was fractionated onto a
15 to 60% Suc gradient. RNA was isolated from each of the fractions
and prolamine mRNA quantified by dot-blot analysis. Black bars,
Soluble; hatched bars, monosome; and shaded bars, polysome.
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Isolation of PB Populations
The resistance of the prolamine polysome-PB interaction to Triton
X-100 detergent treatment but sensitivity to solutions of high ionic
strength suggests that the prolamine polysomes may be anchored to the
cytoskeleton. This type of detergent-resistant, salt-sensitive
interaction exists for other cytoskeleton-bound polysomes (Davies et
al., 1991, 1993; Pachter, 1992). To better characterize the specific
interaction of the prolamine polysomes with the C-ER and PB-ER
membranes and their possible interaction with the cytoskeleton,
experiments were initiated to isolate enriched fractions of these two
membrane types. Mid-developing rice seeds were ground in CSB, filtered
through Miracloth, and layered onto a 20 to 80% Suc gradient and
centrifuged at 300,000g for 60 min. Four major peaks were
visible on the UV absorbance profile (Fig. 5), and consisted of: an upper peak
corresponding to the soluble protein fraction (fraction 1); a narrow,
second peak corresponding to the 80S monosome fraction (fraction 2); a
broad, third peak (fractions 3 and 4); and a narrow, fourth peak
(fraction 5).
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| Figure 5.
Suc-gradient (20-80%) profile of crude rice seed
tissue extracted in CSB. One gram of seed was extracted in 3 mL of CSB
and 250 µL of this was layered onto the gradient. The gradient was separated into six fractions, the last fraction corresponding to the
dense pellet at the bottom of the gradient. Solid bars represent the
percent prolamine mRNA content and dashed bars represent the percent
glutelin mRNA content in each of the fractions as determined by
dot-blot analysis.
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The positions of peaks 3 and 4 in the profile initially suggested that
they corresponded to the C-ER and PB-ER, respectively, because the
dense PBs would sediment lower in the gradient. Biochemical evidence,
however, indicates that peaks 3 and 4 were qualitatively similar in
composition; they both contained abundant levels of prolamine mRNAs in
approximately equal amounts (Fig. 5). Prolamine polypeptides
co-fractionated with prolamine mRNAs and were readily detectable in
fractions corresponding to peak 3, although they were most abundant in
peak 4 (see Fig. 7, A and B, top panels). In addition, glutelin mRNAs
were not enriched in fractions corresponding to peak 3, but were found
in peak 4 (Fig. 5). Phase-contrast microscopy of peaks 3 and 4 demonstrate that the PBs in these peaks differed in their size and
abundance (Fig. 6). Peak 3 contained
fewer, smaller PBs compared with the larger, more abundant PBs of peak 4. Overall, these observations indicate that peaks 3 and 4 are membrane
fractions consisting of a complex of C-ER and PB-ER, which appear
distinguishable by the size and abundance of the PBs. Peaks 3 and 4, therefore, will be referred to as light and heavy membrane fractions,
respectively.
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| Figure 7.
Suc-density gradient UV-absorbance profiles of
light (A) and heavy (B) membrane fractions. Light and heavy membrane
fractions were isolated onto a 20 to 80% Suc-density gradient, divided
into three aliquots, and then the aliquots were treated with CSB (top panels), CSB plus 1% PTE detergent (middle panels), or
high-ionic-strength detergent solution (buffer U, bottom panels). These
were then refractionated onto 20 to 80% Suc gradients as shown here.
The numbers and letters at the bottom of each panel are fractions of
the Suc-density gradient that correspond to soluble fraction (1), light
membrane fraction (2), heavy membrane fraction (3), and pellet fraction
(P). Bars represent percent prolamine mRNA content as in Figure 5.
Below each Suc-density gradient profile are immunoblots of protein
extracted from each fraction. Blots were probed with
anti-prolamine (prol), anti-actin (act), and anti-tubulin (tub)
antibodies.
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| Figure 6.
Phase-contrast microscopy of peak 3 (A, the light
membrane fraction) and peak 4 (B, the heavy membrane fraction) showing
representative complexes of membranes and PBs. Arrows indicate the
smaller, less-abundant PBs among membranes in peak 3. Bar = 10 µm.
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The Association of Prolamine mRNA, ER Membranes, and the
Cytoskeleton
The light and heavy membrane fractions were isolated and
individually incubated in low-ionic-strength CSB, CSB plus 1% PTE (a
nonionic detergent), or high-ionic-strength buffer U at 4°C for 10 min, and then refractionated through a 20 to 80% Suc-density gradient
(Fig. 7). The profiles of these gradients
were divided into four fractions: fraction 1 corresponds to the
supernatant; fraction 2 corresponds to the light membrane fraction;
fraction 3 corresponds to the heavy membrane fraction; and fraction 4 (pellet fraction) consists of the dense pellet that was resuspended
from the bottom of the centrifuge tube and, therefore, does not have a
measured absorbance (Fig. 7). Both the light and heavy membrane fractions in CSB sedimented as expected when compared with their profile positions in Figure 5 (Fig. 7, A and B, top panels). Dot-blot hybridization and protein gel-blot analysis showed that the
majority of prolamine mRNA, prolamine protein, actin, and tubulin
co-fractionated with the light and heavy membrane fractions (Fig. 7, A
and B, top panels). When the light membrane fraction was incubated in CSB plus 1% PTE to dissolve the membranes and then refractionated on
the Suc-density gradient, the sedimentation profile changed markedly.
The major absorbance peak increased in buoyant density and banded at
the bottom of the gradient near the heavy membrane fraction (Fig. 7A,
middle panel). Most of the prolamine mRNA, prolamine protein, actin,
and tubulin was found to be associated with the densely sedimenting
absorbance peak (Fig. 7A, middle panel), although there was a
significant amount of mRNA and protein that pelleted to the bottom of
the gradient. When the heavy membrane fraction was detergent treated
and refractionated in the same manner, the major peak sedimented toward
the bottom of the gradient with a slight increase in density compared
with the peak observed in CSB alone (Fig. 7B, middle panel). Much of
the prolamine mRNA, prolamine protein, actin, and tubulin co-sedimented
with this major absorbance peak (Fig. 7B, middle panel).
Treatment of both the light and heavy membrane fractions with buffer U
resulted in the release of monosomes and polysomes (Fig. 7, A and B,
bottom panels). Prolamine mRNA was most abundant in the
monosome/polysome fraction (fraction 2), whereas prolamine protein
remained primarily associated with the heavy membrane fraction
(fraction 3) in both profiles. Buffer U treatment of both the light and
heavy fractions solubilized much of the actin and tubulin. However,
some of these proteins were resistant to this treatment and remained in
the lower fractions (Fig. 7, A and B, bottom panels). These experiments
indicate that most of the prolamine polysomes are complexed not only
with the ER membranes but with the cytoskeleton as well. In addition,
because solubilization of the membrane with nonionic detergent did not
release the prolamine mRNA, this would suggest that the prolamine
polysomes are bound to the cytoskeleton, either directly or indirectly.
It is interesting that glutelin polysomes behaved in a manner similar
to prolamine polysomes under these conditions (data not shown),
suggesting that a common association may exist between the prolamine
and glutelin polysomes and their respective ER domains.
The Effect of Cytoskeleton Inhibitors on the Association of
Prolamine mRNA and PBs
In an effort to determine if there is a functional association of
the cytoskeleton with prolamine mRNA, the effects of
cytoskeleton-destabilizing drugs on polysome release were studied.
Cytochalasin B and nocodazole were used to destabilize microfilaments
and microtubules, respectively. Panicles bearing mid-developing seeds
were immersed in nutrient solution alone or solution containing 100 µm cytochalasin B or 100 µm nocodazole for
30 h. Seeds were ground in buffer U to solubilize the membranes
and cytoskeleton, and the extract was fractionated on a polysome
gradient. The polysome profiles of control and cytochalasin-treated seed extracts are shown in Figure 8. The
cytochalasin B-treated seed profile showed a reduced amount of
polysomes compared with the control seeds. However, no significant
decrease in prolamine mRNA in the cytochalasin B-treated versus the
control samples was observed (Fig. 8).
View larger version (20K):
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| Figure 8.
Suc-gradient profiles from control seeds (A) or
seeds treated with 100 µm cytochalasin B (B) for 30 h. Seeds (500 mg) were ground in 2 mL of buffer U and filtered before
layering 200 µL onto a 15 to 60% gradient. Bars represent relative
prolamine concentrations as determined from dot-blot scans.
|
|
To determine if a certain percentage of prolamine polysomes in the
cytochalasin-treated seeds were in fact released from the cytoskeleton
but not immediately degraded, we determined the prolamine mRNA content
in a PB/cytoskeleton fraction to eliminate any free polysomes. Control
and cytochalasin B-treated seeds were extracted in CSB plus 1% Triton
X-100 and the extract was fractionated through a 20 to 80% Suc
gradient. The major absorbance peak contained the PB/cytoskeleton
fraction. Total RNA was isolated from this peak from both the control
and the cytochalasin B-treated samples. Dot-blot analysis using a
prolamine probe showed that there was only 3.5 ± 2% less
prolamine mRNA associated with this fraction in cytochalasin B-treated
than in control plants. Similar results were seen when 100 µm
cytochalasin D was used in place of cytochalasin B, and when
half-sectioned seeds were treated for 6 h with these cytoskeleton-destabilizing drugs rather than panicles. These results suggest that the microfilament network that is associated with the PB
surface is not involved in anchoring prolamine polysomes or mRNA, or
that this network is resistant to the destabilizing effects of
cytochalasin. Nocodazole treatment showed a very similar profile and
prolamine mRNA content in each of the fractions compared with the
control (data not shown), suggesting that the microtubule component of
the cytoskeleton does not play a role in binding of cellular polysomes
to the cytoskeleton.
| |
DISCUSSION |
The association of mRNAs and polysomes with the cytoskeleton has
been well documented in several animal cell types, including Drosophila and Xenopus spp. oocytes, and certain
types of cultured cells such as HeLa, fibroblasts, and oligodendrocytes
(for review, see Hesketh, 1994; St. Johnston, 1995). One of the
functions of this interaction is to localize mRNAs as a mechanism for
achieving translation in specific locations within the cell. In plants
the association of polysomes with the cytoskeleton has been
demonstrated in pea roots and stems and in maize endosperm (Davies and
Abe, 1989; Davies et al., 1993; Ito et al., 1994). The microfilament component of the cytoskeleton in maize endosperm cells is largely concentrated in spheres surrounding the PB (Abe et al., 1991; Clore et
al., 1996). Polysomes are localized to this cytoskeleton and are
released by solutions of high ionic strength and other reagents that
depolymerize actin, but not PTE (Davies et al., 1993). The percentage
of release of polysomes from the maize PBs by increasing concentrations
of salt is proportional to the number of ribosomes associated with the
polysome, suggesting that the ribosome anchors the polysome to the
cytoskeleton (Davies et al., 1993). We show that, as in maize, in rice
endosperm PB fractions most of the polysomes remain associated with the
cytoskeleton after extraction in nonionic detergent and are
subsequently released after treatment with high-ionic-strength
detergent (Figs. 2 and 7). These data suggest that F-actin seems to be
the scaffold upon which other components (i.e. membranes, microtubules,
and polysomes) are associated (Davies et al., 1993). Our results differ
from those seen in maize with respect to the type of interaction of the
prolamine polysomes with the cytoskeleton. The results of the NaF
treatments (Figs. 3 and 4) demonstrate that the prolamine mRNA
cytoskeleton interaction in rice endosperm is mediated by the RNA, but
do not rule out the additional role of ribosomes in this interaction.
The ribosome-free prolamine mRNA appears to bind more tightly to the
cytoskeleton than do polysomes because higher concentrations of salt
are required to release these ribosome-free mRNAs (Fig. 4). In animal
systems there is evidence for the association of the mRNA and the
ribosome with the cytoskeleton, and it is suggested that both have an
anchoring role (Hesketh, 1994).
Crude rice endosperm extracts can be resolved into two membrane
fractions on Suc-density gradients. The buoyant densities of these two
membrane fractions (peaks 3 and 4; Fig. 5) initially led us to believe
that the C-ER membranes were enriched in peak 3 and the PB-ER membranes
were enriched in peak 4. However, the presence of equal amounts of
prolamine and glutelin mRNAs in each of the fractions suggests that
both the C-ER and PB-ER membranes were present in both fractions, since
our previous work demonstrated that glutelin mRNA is enriched on the
C-ER and prolamine mRNA is enriched on the PB-ER (Li et al., 1993a).
The distinguishing feature between these two fractions is that they
differ in PB size and abundance (Fig. 6). The fact that both membrane
fractions are associated with the cytoskeleton (Fig. 7) may provide an
explanation for the finding that both ER types are found in the light
and heavy membrane fractions. As shown previously in maize, F-actin appears to hold zein PBs into aggregates in crude extracts prepared in
CSB (Abe et al., 1991). In rice F-actin also surrounds the prolamine
PBs with longer, less densely staining filaments running throughout the
cytoplasm (D.G. Muench and T.W. Okita, unpublished data). Perhaps this
cytoskeleton network also associates with the C-ER network, thus
forming a C-ER/PB-ER complex held together by the cytoskeleton.
Complexes containing large PBs would then have a greater density and
would sediment to the lower membrane fraction, whereas those ER
membranes having very small, developing PBs, or none at all, would be
less dense and would sediment to the light membrane fraction. The
membrane component of the light membrane fraction is responsible for
its buoyancy, since treatment of this fraction with PTE resulted in a
dramatic shift to the lower portion of the gradient (Fig. 7A, top and
middle panels). The large PBs in the heavy membrane fraction counter
the buoyancy of the membrane in this fraction; however, upon PTE
treatment there was a small shift in the sedimentation of this peak
(Fig. 7B, top and middle panels).
Several researchers have shown that fragments of insolubilized ER
membranes remain after cells are treated with nonionic detergent (Dang
et al., 1983; Hesketh and Pryme, 1991; Vedeler et al., 1991). These
insolubilized membrane remnants may be responsible for associating membrane-bound polysomes with the detergent-resistant fraction, even
though these polysomes may not be associated with the cytoskeleton. Other researchers claim that nonionic detergents adequately dissolve the membrane and allow for the release of membrane-bound polysomes with
high-ionic-strength solutions (Lenk et al., 1977; Adams et al., 1983;
Fey et al., 1986). For example, maize PB fractions treated with
nonionic detergent completely dissolved the membrane, as determined by
lipid analysis and loss of fluorescent membrane staining (Abe et al.,
1991; Ito et al., 1994). We have shown that Triton X-100 is very
efficient in solubilizing the membrane and membrane-associated proteins
in the PB pellet (Table I; Fig. 1). Although it is possible that a very
small amount of membrane remains in our extracts, the fact that
ribosome-free prolamine mRNAs are still associated with the
cytoskeleton (Fig. 3) demonstrates that ribosome-membrane interactions
are not required for the anchoring of prolamine mRNA to the PB.
Cytochalasin B treatment caused a reduction in the amount of total
polysomes in the cell; however, it does not release prolamine mRNA from
the cytoskeleton (Fig. 8). Although this was an unexpected result, it
may be that prolamine mRNA is bound to a population of F-actin that has
some resistance to the effects of cytochalasin treatment. When rice
endosperm sections are treated with cytochalasin B or D and then
F-actin is visualized by Texas red-phalloidin staining, there is no
visible effect on the quantity or morphology of the actin around the
PBs, even though the cells showed actin aggregation around the nucleus
(D.G. Muench and T.W. Okita, unpublished data), which is a common
result of cytochalasin treatment (Spector et al., 1989). This result
differs from what was seen in maize, in which treatment of endosperm
sections with cytochalasin D resulted in obvious changes in actin
organization when visualized by indirect immunofluorescence (Clore et
al., 1996).
Alternatively, it may be that prolamine mRNAs are not complexed with
actin or tubulin, but with a different component of the ER-associated
cytoskeleton. An example of an mRNA-cytoskeleton interaction such as
this was shown by a specific membrane-bound mRNA in HeLa cells
(Zambetti et al., 1990a). In that study cytochalasin D and puromycin
together did not release the mRNA from the membrane. Because
microtubules did not fractionate with these membranes, the authors
suggested that another component of the cytoskeleton is binding the
mRNAs, perhaps the intermediate filaments, and they raised the
possibility that mRNAs are associated with the cytoskeleton in a
heterogeneous manner. Results from other studies are also consistent
with this view. In Fucus spp. actin mRNA is localized to the
cell plate in dividing embryos; however, drugs that affect the
integrity of the cytoskeleton do not affect the localization of the
actin mRNA (Bouget et al., 1996). Also, microfilament-destabilizing drugs did not affect the association of actin mRNA and histone mRNA
with the cytoskeleton in ascidian eggs (Jeffery, 1984).
Okita et al. (1994) proposed three possible models for the nonrandom
localization of rice storage protein mRNAs to the ER in endosperm
cells. One model suggests that there are different populations of
signal-recognition particles that associate with specific subdomains of
the ER, so that prolamine and glutelin mRNAs are docked to different
domains via their encoded signal peptides. The second model proposes
that the long-lasting interaction of BiP to the nascent and mature
prolamine polypeptide and to the prolamine PB (Li et al., 1993b; Muench
et al., 1997) could enrich prolamine mRNAs around the PB because of a
longer retention time on the PB-ER. The third model proposes a direct
RNA-sorting mechanism as a result of an RNA signal that would
specifically target prolamine mRNA to the PB-ER in association with the
cytoskeleton (Okita et al., 1994). Although we cannot argue for or
against any of these mechanisms as being responsible for the enrichment of prolamine mRNA to the PB-ER, it appears that this mRNA associates with the cytoskeleton that surrounds the prolamine PBs. Because glutelin polysomes are also cytoskeleton bound, the anchoring mechanism
on the surface of the ER may be the same between these two polysome
types. Therefore, the differential sorting of prolamine and glutelin
mRNAs may occur before their anchoring at the surface of the respective
ER membranes. The fact that ribosome-free prolamine mRNA retains the
ability to bind to the cytoskeleton suggests that it may be localized
before translation in a manner similar to that by which transcripts
such as bicoid or oskar are localized in Drosophila spp., or
myelin basic protein are localized in oligodendrocytes (St. Johnston,
1995). Our current efforts are focused on determining the mechanism
that mediates the localization of storage protein mRNAs in rice.
| |
FOOTNOTES |
1
This work was supported by U.S. Department of
Agriculture National Research Initiative Competitive Grants Program
Award no. 94-37304-1174 to T.W.O.
2
D.G.M. and Y.W. contributed equally to this
publication.
3
Present address: Department of Biological
Sciences, 2500 University Drive N.W., University of Calgary, Calgary,
AB, Canada, T2N 1N4.
*
Corresponding author; e-mail tokita{at}wsu.edu; fax
1-509-335-7643.
Received July 14, 1997;
accepted October 26, 1997.
| |
ABBREVIATIONS |
Abbreviations:
C-ER, cisternal ER.
CSB, cytoskeleton-stabilizing buffer.
FA, fatty acid.
PB, protein body.
PTE, polyoxyethylene 10-tridecyl.
X:Y, a fatty acyl group containing X
carbon atoms and Y cis double bonds.
| |
ACKNOWLEDGMENT |
We thank Dr. Anders Carlsson for his assistance with the FA
analysis.
| |
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Top
Abstract
Introduction