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Plant Physiol. (1998) 116: 1339-1350
Cloning and Characterization of AtRGP11
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.
The primary cell wall of dicot plants is laid down by young cells
prior to the cessation of elongation and secondary wall deposition.
Making up to 90% of the cell's dry weight, the extracellular matrix
is important for many processes, including morphogenesis, growth,
disease resistance, recognition, signaling, digestibility, nutrition,
and decay. The composition of the cell wall has been extensively
described (Bacic et al., 1988 Heteropolysaccharide biosynthesis can be divided into four steps: (a)
chain or backbone initiation, (b) elongation, (c) side-chain addition,
and (d) termination and extracellular deposition (Waldron and Brett,
1985 The enzymes involved in wall biosynthesis have been recalcitrant to
isolation (Carpita et al., 1996 During studies of polysaccharide synthesis in pea (Pisum
sativum) Golgi membranes, Dhugga et al. (1991) We were interested in studying various aspects of cell wall metabolism,
including the synthesis of polysaccharides and their delivery to the
cell wall. Studies in pea have shown that a 41-kD protein may be
involved in cell wall polysaccharide synthesis, possibly that of
xyloglucan (Dhugga et al., 1997 Plant and Cell Growth
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Levy and Staehelin, 1992; Zablackis et
al., 1995), and yet many questions remain unanswered regarding the
synthesis and interaction of these components to provide cells with a
functional wall (Carpita and Gibeaut, 1993; Carpita et al., 1996).
). The similarity between various polysaccharide backbones leads to
the prediction that the synthesizing machinery would be conserved
between them. For example, the backbone of xyloglucan polymers, 
-1,4
glucan, can be synthesized independently of or concurrently with
side-chain addition (Campbell et al., 1988; White et al., 1993), and
this polymer and the chains that make up cellulose are identical. The
later addition of side chains to xyloglucan are catalyzed by specific
transferases (Kleene and Berger, 1993) such as xylosyltransferase
(Campbell et al., 1988), galactosyltransferase, and fucosyltransferase
(Faïk et al., 1997), all of which are localized to the Golgi
compartment (Brummell et al., 1990; Driouich et al., 1993; Staehelin
and Moore, 1995).
; Albersheim et al., 1997). Only
recently has the first gene encoding putative cellulose biosynthetic enzymes, celA, been isolated from cotton (Gossypium
hirsutum) and rice (Oryza sativa; Pear et al.,
1996).
identified a 41-kD protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-Glc but that this labeling could be reversibly competed with by unlabeled UDP-Glc, UDP-Xyl, and UDP-Gal, the sugars
that make up xyloglucan (Hayashi, 1989). The 41-kD protein was named
PsRGP1 (P.
sativum Reversibly
Glycosylated Polypeptide-1; Dhugga et al.,
1997). Furthermore, the conditions that stimulate or inhibit
Golgi-localized -glucan synthase activity are the same conditions
that stimulate or inhibit the glycosylation of PsRGP1 (Dhugga et al.,
1991). To address the role of this protein in polysaccharide synthesis,
the authors purified the polypeptides and obtained the sequences from
tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against
PsRGP1 showed that it is soluble and localized to the plasma membrane
(Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In
addition to its Golgi localization, the steady-state glycosylation of
PsRGP1 is approximately 10:7:3 (UDP-Glc:-Xyl:-Gal), which is similar to
the typical sugar composition of xyloglucan (1.0:0.75:0.25; Dhugga et
al., 1997).
). Here we report the characterization
of AtRGP1 (Arabidopsis
thaliana Reversibly
Glycosylated Polypeptide-1), a soluble protein
that can also be found weakly associated with membrane fractions, most likely the Golgi fraction. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make
polysaccharides (nucleotide sugars) suggests a possible role for AtRGP1
in polysaccharide biosynthesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
1, 10 g L1 Suc,
4.3 g L1 Murashige-Skoog salts
[GIBCO-BRL], 0.5 g L1 Mes, 0.1 g
L1 myo-inositol, 1 mg
L1 thiamine-HCl, and 0.5 mg
L1 nicotinic acid, pH 5.7). Plants grown in
liquid culture and maintained under constant agitation (50-60 rpm) and
light grew mostly roots.
and were maintained by diluting a 2-week-old culture 1:5 with fresh suspension-culture medium (3.2 g
L1 Gamborg's B5 medium [Sigma], 20 g
L1 Suc, 2.5 µm 2,4-D, and
0.5 g L1 Mes, pH 5.7).
Cloning, Sequencing, and Sequence Analysis
Peptide sequences from purified pea (Pisum sativum) proteins (Dhugga and Ray, 1994; Fig. 1)
were used to search the EST database (dBEST) comprising the GenBank,
EMBL, and DDBJ databases. We used a database search program (Basic
BLAST) with the "tblastn" option, which compares a protein query
sequence against a nucleotide sequence database dynamically translated
in all reading frames (Altschul et al., 1990,
http://www.ncbi.nlm.nih.gov/BLAST/). Twenty-eight ESTs have been
obtained so far from the original search and subsequent searches
(Altschul et al., 1997) using the ESTs themselves to find overlapping
ESTs. These ESTs can be grouped based on sequence similarity using a
software package (MegAlign module, DNAStar, Genetics Computer
Group, Madison, WI).
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) kept by the Arabidopsis Biological Research Center at Ohio State University (Columbus). Automated sequencing of the
cDNAs was performed by the Plant Biochemistry Facility at Michigan
State University (East Lansing).
. Protein-sequence
analysis of AtRGP1 was also performed with this software package. In
addition, the pSORT program (http://psort.nibb.ac.jp/) was used, which
compares a given protein sequence against a database of known sequences
that mediate protein sorting to various membranes and organelles.
Isolation of RNA and Northern Analysis
Total RNA from Arabidopsis flowers, leaves, roots, and stems was extracted as described previously (Puissant and Houdebine, 1990). Total
Arabidopsis RNA (30 µg/lane) was separated on a gel containing 1%
agarose and 2% formaldehyde. Electrophoresis was stopped when the dye
front had migrated three-quarters of the way down the gel. Sizes were
estimated using the sizes of rRNAs as markers. Gels were washed for 20 min in 10× SSC and analyzed as described by Sambrook et al. (1992).
Transfer was performed by capillary action onto a nylon membrane
(Hybond N, Amersham) overnight. Membranes were washed in 2× SSC for 5 min, cross-linked in a UV-cross-linker, and stored at room temperature
between filter papers. Hybridization was overnight at 65°C in
northern hybridization buffer (5× SSC, 10× Denhardt's solution [2
mg mL1 Ficoll, 2 mg mL1
PVP, 2 mg mL1 BSA], 0.1 m
KPO4, pH 6.8, 100 µg/mL salmon-sperm DNA
[freshly boiled], 10% dextran, and 30% deionized formamide) using a
random-primed 32P-labeled 1.0-kb
EcoRI-BbvI fragment of the AtRGP1 cDNA at
1,000,000 dpm mL1. Membranes were washed once
with 2× SSC and 0.5% SDS, twice with 2× SSC and 0.1% SDS, and once
with 0.2× SSC and 0.1% SDS (1× SSC is 0.15 m NaCl and 15 mm sodium nitrate, pH 7.0), each for 30 min at 65°C prior
to autoradiographic exposure.
Protoplast Preparation
Twenty grams of cells from a 5- to 8-d-old cell-suspension culture collected on an 80-µm filter was incubated with 50 mL of freshly made protoplasting solution (15.4% [w/v] Suc, 0.32% [w/v] Gamborg's B5 minimal organic, 5 mg mL1
cellulase [Onozuka R10, Yakutt Honsha, Tokyo], and 1.2 mg
mL1 Macerozyme R10 [Yakutt Honsha]) for
2 h in a rotary shaker at 80 rpm. All manipulations were done at
room temperature. The cells were then poured into Babcock centrifuge
bottles and centrifuged for 10 min at 1100 rpm in a clinical centrifuge
swinging bucket rotor (model HNSII, IEC, Needham Heights, MA). Floated
protoplasts were collected, mixed with 20 mL of 0.4 m
betaine, 3 mm Mes, and 10 mm
CaCl2, pH 5.7, and then pelleted for 5 min at
50g.
Protein Extraction
Four milliliters of cold lysis buffer (20 mm Hepes-KOH, pH 7.0, 13.5% [w/v] Suc, 10 mm potassium acetate, 1 mm DDT, 0.5 mm PMSF, and 1 mm EDTA) was added per milliliter of packed protoplasts and passed through a 25-5/8-gauge needle at 4°C until no unbroken protoplasts could be detected under the microscope. Cell debris were pelleted by centrifugation at 500g for 5 min and this homogenate was called the total protoplast protein. Total plant protein from different tissues (flowers, leaves, roots, stems, and roots grown in liquid culture) was extracted by grinding 1 to 5 g of the given tissue in liquid N2 and mixing with 5 mL of lysis buffer at 4°C. For all plant tissue samples, cell debris were pelleted by centrifugation at 500g for 5 min. This homogenate was called the total tissue protein, where "tissue" specifies the tissue used.Overexpression of a Fusion Protein in Escherichia coli and Antibody Preparation
The 1.4-kb EcoRI-NotI fragment of AtRGP1 (GenBank accession no. AF013627), encoding all but the first nine amino acids from the 5
end of the cDNA, was cloned into
the EcoRI-NotI sites of pGEX-5X-2 (Pharmacia) to
generate an N-terminal in-frame fusion with the 26-kD domain of GST.
This fusion was overexpressed in E. coli (strain DH5
) by
growing cells in Luria-Bertani medium at 37°C to an
A600 of 0.7 to 0.8, then adding
isopropylthio--d-galactosidase to a final
concentration of 0.2 mm, and incubating the culture at
28°C for 4 h. The soluble GST fusion protein was purified as described previously (Bar-Peled and Raikhel, 1996). Five hundred microliters (0.4-0.5 mg) of eluted GST-AtRGP1 fusion protein was emulsified by sonication with an equal amount of TiterMax adjuvant (CytRx Corp., Norcross, GA) and injected into New Zealand White rabbits. An equal amount of GST-AtRGP1 protein was injected into the
rabbits two more times to boost the immune response over a 2-month
period. Blood was taken from the rabbits 1 month after the first
injection and every other week thereafter and treated as described
previously (Harlow and Lane, 1988).
Timed Studies of RNA and Protein Levels
A 0.5-L Arabidopsis cell-suspension culture was started by adding 50 mL of a 7-d-old culture to 450 mL of fresh suspension-culture medium. Fifteen-milliliter aliquots were taken at 24-h intervals over a 12-d span. Each fraction was centrifuged at 50g for 5 min, the supernatant was removed, and the weight of the packed cells was measured. Equal amounts of protein (50 µg/lane) were separated by SDS-PAGE (Laemmli et al., 1970), and the presence of AtRGP1 was determined by immunoblotting. Equal amounts of RNA (30 µg/lane) were separated in a 1% agarose and 2% formaldehyde gel, and the level of AtRGP1 RNA was determined by northern analysis.Reaction with UDP-Sugars
One hundred to 250 µg of total Arabidopsis, pea, tobacco, or maize total protein from roots was labeled as described previously (Dhugga et al., 1991). First, 0.1 µCi of
UDP-d-[U-14C]Glc (specific activity
263 µCi µmol1 [ICN]) or 0.5 µCi of
UDP-d-[6-3H]Gal (specific activity
10.6 mCi µmol1 [ICN]), was added to the
protein sample in a volume of 50 to 150 µL of lysis buffer containing
3 mm MgCl2. The reactions were stopped after a 10-min incubation at room temperature by adding 10 to
30 µL of SDS-PAGE loading buffer (120 mm Tris, pH 6.8, 200 mm DTT, 4% SDS, 0.02% bromphenol blue, and 20%
glycerol). The protein samples were separated by SDS-PAGE and treated
as described below. Since the UDP-Glc and UDP-Gal reactions gave the
same results, only the UDP-Glc reactions are presented.
1; Sigma), UDP, Glc, UDP-Glc, UDP-Xyl,
UDP-Gal, or UDP-Man was added to a final concentration of 3 mm and the reactions continued for 10 more min. To stop the
reactions, SDS-PAGE loading buffer (120 mm Tris, pH 6.8, 200 mm DTT, 4% SDS, 0.02% bromphenol blue, and 20%
glycerol) was added. The protein samples were separated by SDS-PAGE and
treated as described below.
Immunochemical Studies
Immunoprecipitation
Immunoprecipitations were as described previously (Harlow and Lane, 1988). Anti-AtRGP1 polyclonal antibodies were cross-linked to
protein A-Sepharose 6MB (Pharmacia) and mixed with total protein from
roots grown in liquid culture. After the samples were washed three
times with PBS and once with 10 mm potassium phosphate
buffer, pH 7.0, proteins bound to the antibodies were eluted
with 100 mm Gly-HCl, pH 2.5. Protoplasts labeled with
[35S]Met were incubated overnight in a rotary
shaker at 80 rpm, and total protein was prepared as described
above, and immunoprecipitated.
SDS-PAGE and Immunoblotting
Protein concentration was determined as described previously (Bradford, 1976) using BSA as the standard. Protein molecular weight
standards were purchased from Bio-Rad (low- and broad-range markers,
catalog nos. 161-0304 and 161-0317, respectively). All protein samples
were separated on 12.5% SDS-PAGE gels using 1× running buffer (250 mm Tris, 1 m Gly, and 0.5% SDS).
80°C prior to film development.
2 for 2 h. Filters were stained
with 1× Ponceau-S (2% Ponceau-S, 30% TCA, and 30% sulfosalicylic
acid) and blocked overnight in 10% (w/v) dry milk powder in PBST (1×
PBS and 0.1% Tween 20). AtRGP1 polyclonal antibodies were used at a
1:1000 dilution. Anti-RD28 plasma membrane marker sera was used at
1:500 dilution and anti-binding protein ER lumen protein at 1:1000
dilution. They were a gift from Maarten Chrispeels (University of
California, San Diego). Anti-AtELP (partially Golgi-localized protein,
Ahmed et al., 1997) and anti-AtPEP12p (post-Golgi compartment protein,
Conceição et al., 1997) polyclonal antibodies were used at
a 1:500 dilution. ARA4 monoclonal antibody (a Golgi membrane-localized
and -soluble protein, Ueda et al., 1996) was used at 1:500 dilution.
1 final concentration)
and nitroblue tetrazolium (300 µg mL1 final
concentration) in alkaline buffer.
Localization
Differential Centrifugation
Total protoplast protein was centrifuged at 1,000g for 20 min at 4°C. The pellet (p1) was washed with lysis buffer and centrifuged again. The supernatant (s1) was centrifuged at 5,000g for 30 min. The pellet was washed with lysis buffer and centrifuged again (now called p5). The supernatant (now s5) was treated in the same way for sequential differential centrifugations at 15,000, 25,000, 50,000, and 100,000g, with the last two centrifugations carried out for 40 min. All pellets (p1, p5, p15, p25, p50, and p100) were resuspended in lysis buffer and the protein was quantified.Membrane Association
Total microsomes were prepared by centrifugation of total protoplast protein at 150,000g for 1 h and washing the pellet with lysis buffer. Pellets were resuspended with lysis buffer (total protein) or lysis buffer containing 0.1, 0.5, or 1.0% Triton X-100, 0.5 or 2 m urea, 0.1 m sodium carbonate, 0.1, 0.5, or 1.0 m NaCl, or 0.1 m potassium phosphate, pH 7.0, for 1 h on ice, and centrifuged again at 150,000g for 1 h. The resulting pellets were washed with lysis buffer, resuspended in loading buffer, and separated by SDS-PAGE prior to analysis by immunoblot using anti-AtRGP1 antibodies.Suc-Density-Gradient Centrifugation
Total protoplast protein was centrifuged at 1000g for 10 min at 4°C, and 6 mL of the supernatant (S1) was loaded on top of a 16 to 55% equilibrium-density Suc gradient modified from Gibeaut and Carpita (1994). To prepare the gradient, stock solutions of 55, 40, 33.5, 26.5, and 16% (w/v) Suc buffered in 10 mm Hepes-KOH, pH 7.0, 10 mm potassium acetate, and 2 mm EDTA
were prepared. Gradients were prepared by the sequential addition of
2.7 mL of 55% Suc solution, 8.8 mL of 40% Suc solution, 7.0 mL of
33.5% Suc solution, 6.0 mL of 26.5% Suc solution, and 4.5 mL of 16% Suc solution to 35-mL polyallomer tubes (catalog no. 326823, Beckman). After these solutions were carefully layered, they were centrifuged at
150,000g for 18 to 20 h at 4°C in a rotor (model SW28, Beckman). This resulted in an equilibrium-density gradient similar to one obtained using a gradient maker. Equal-volume fractions were collected from the bottom of the gradients, and the Suc concentration was estimated by measuring the refractive index of the individual fractions
using a refractometer. Aliquots of the fractions (50-100 µL) were
separated by SDS-PAGE, and the separation of AtRGP1 and membrane marker
proteins were analyzed by immunoblotting.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Identification of an Arabidopsis cDNA Encoding an AtRGP1 Protein
Sequences from three purified tryptic peptides from the pea RGP protein doublet were presented by Dhugga and Ray (1994) and used to
search the dBEST database. A total of 28 Arabidopsis ESTs have been
obtained so far from the original and subsequent searches. Based on
sequence similarity, these ESTs can be assigned to at least three
different groups, denoted AtRGP1 (AF013627),
AtRGP2 (AF013628), and AtRGP3 (AF034255). These
cDNAs share between 93 and 99% sequence identity at the amino acid
level. Because the similarity is spread through the whole sequence,
only one cDNA, AtRGP1, was used for further
analysis (Fig. 1A).
) predicts that
AtRGP1 is a hydrophilic protein (data not shown). Visual and
computer-aided (pSORT, http://psort. nibb.ac.jp/) analyses of the
protein sequence predict that the protein lacks a signal sequence that
might direct AtRGP1 to a membrane compartment within the cell. The same
computer program predicts the existence of an
N-myristoylation site in AtRGP1 (Fig. 1A).
N-myristoylation has been shown to aid in the association of
otherwise soluble proteins to the cytosolic surface of membranes.
-glycosyl residues from either
UDP-Glc or UDP-GlcNAc (Saxena et al., 1995), as well as UDP-Glc-binding
proteins (Delmer and Amor, 1995; Pear et al., 1996). AtRGP1 is 40%
identical and 53% similar to the predicted UDP-Glc-binding domain (U1)
of cotton CelA (Delmer and Amor, 1995; Pear et al., 1996; Saxena
and Brown, 1997). Furthermore, AtRGP1 contained the critical Asp
residue (Fig. 1B). In contrast, sequence identity between the deduced
amino acid sequence of cotton CelA and AtRGP1 is very low,
approximately 15%.
demonstrated that the Glc from UDP-Glc was attached
to an Arg residue of a maize protein with high sequence similarity to
AtRGP1. The tryptic peptide containing the glucosylated Arg is 93%
identical to a region of AtRGP1 (Fig. 1A). Dhugga et al. (1997) noted
this same similarity to PsRGP1 and postulated that Arg-158 is the
location of a sugar addition in the pea protein. Similarly, we
postulate that Arg-158 is the location of Glc addition in the
Arabidopsis protein.
Distribution of RGP1 Transcript and Protein
The isolation of RGP proteins in both monocots and dicots suggests a general function for this protein. To confirm the presence of proteins similar to RGP, as evidenced by database searches, we used AtRGP1 antibodies in immunoblots against protein from various organisms. Immunoreactive polypeptides of 41 kD were detected in Arabidopsis, pea, tobacco, and maize (Fig. 2). Although pea RGP1 has been referred to as a polypeptide of about 40 kD (Dhugga et al., 1991, 1997), it is
clear from the cloned cDNA that PsRGP1 has a predicted size of 41 kD.
Since all RGPs so far identified encode proteins of approximately the
same size, we will refer to them as 41-kD proteins.
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AtRGP1 Is Soluble and Membrane Associated
Glycosylation of AtRGP1
We have isolated and characterized a cDNA clone that encodes the
Arabidopsis homolog of the PsRGP1 doublet. The PsRGP1 doublet has been
localized to the Golgi compartment and is shown to be reversibly
glycosylated by UDP-Glc. The existence of RGP in both dicots (Dhugga et
al., 1991 Received September 5, 1997;
accepted December 1, 1997.
Abbreviations:
EST, expressed sequence tag.
GST, glutathione
S-transferase.
RGP, reversibly glycosylated
polypeptide.
We thank all of the members of the Raikhel group, especially
Anton Sanderfoot and Diane Bassham, for their help with technical and
conceptual matters; the members of the cell wall group, including Hans
Kende and Jonathan Walton, for helpful discussions; Maarten Chrispeels
for the gifts of BiP and RD 28 antibodies; and Takashi Ueda for ARA-4
antibodies.
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Figure 3.
AtRGP1 RNA and protein are highest in
suspension-cultured cells and roots from whole plants. A, RNA levels in
different tissues. Total RNA (30 µg) from A. thaliana
flowers (lane 1), leaves (lane 2), roots (lane 3), stems (lane 4), and
suspension-cultured cells (lane 5) was separated in a 1% agarose and
6% formaldehyde gel, transferred onto a nylon membrane, and hybridized
with a random-primed, 32P-labeled, 1.0-kb
EcoRI-BbvI fragment of the
AtRGP1 cDNA. The single band obtained is of the expected
size. Molecular mass is indicated in kilodaltons. B, Protein levels in
different tissues. Immunoprecipitations using total protein from
[35S]Met-labeled protoplasts (lane 1) and unlabeled
protoplasts (lane 2) were performed. The former was analyzed by
SDS-PAGE and autoradiography and the latter was analyzed by immunoblot.
Total protein (50 µg) from A. thaliana flowers (lane
3), leaves (lane 4), roots (lane 5), stems (lane 6), root liquid
culture (lane 7), and cell-suspension cultures (lane 8) was analyzed by
immunoblotting using anti-AtRGP1 antibodies. Molecular mass is
indicated in kilodaltons. C, AtRGP1 RNA and protein levels
during the growth cycle of suspension cells. A cell-suspension culture
was started by diluting a 1-week-old culture 10 times with fresh
medium. Samples representing 1/40 of the original volume were collected
at 1-d intervals, and protein and RNA were extracted from each sample.
Top, Total AtRGP1 protein from each sample was analyzed by immunoblot;
bottom, total AtRGP1 RNA was determined as described in
Figure 4A.
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Figure 4.
AtRGP1 is soluble and membrane associated. A,
Total protoplast protein from suspension-cultured cell protoplasts was
centrifuged at 1,000, 5,000, 10,000, 15,000, 25,000, 50,000, and
100,000g. The resultant pellets were resuspended in
lysis buffer and make up fractions p1, p5, p10, p15, p25, p50, and
p100, respectively. s100 denotes the supernatant after the
100,000g centrifugation and represents total soluble
proteins. Equal volumes of protein were separated by SDS-PAGE,
transferred to nitrocellulose, and analyzed by immunoblot. Various
soluble proteins (BiP), integral membrane proteins (AtPEP12p, RD28, and
AtELP), and peripheral membrane proteins (ARA4) were compared with the
membrane association of AtRGP1. The fraction of total protein (T)
present in each pellet is shown. B, AtRGP1 is a peripheral membrane
protein. Total microsomes were prepared by centrifuging total protein
at 150,000g for 1 h and washing the pellet with
lysis buffer. Pellets were resuspended with various buffers for 1 h on ice and centrifuged again at 150,000g for 1 h,
and the pellets were analyzed by immunoblot using anti-AtRGP1 antibodies. Microsomes were resuspended with lysis buffer (lane 1, total protein) or lysis buffer containing 0.1, 0, or 1.0% Triton X-100
(lanes 2, 3, and 4, respectively), 0.5 or 2 m urea (lanes 5 and 6, respectively), 0.1 m sodium carbonate (lane 7), 0.1, 0.5, or 1.0 m NaCl (lanes 8, 9, and 10, respectively), or
0.1 m potassium phosphate buffer, pH 7.0, alone (lane
11).
; Bar-Peled and Raikhel,
1997), and, as expected, BiP shifted to denser fractions. The fact that
AtRGP1 did not shift toward denser fractions in the presence of
Mg2+, as BiP did (data not shown), suggests that
AtRGP1 is not an ER protein.
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Figure 5.
Membrane localization of AtRGP1.
Equilibrium-density gradients were used to fractionate A. thaliana total membrane preparations from suspension-cultured
cell protoplasts. One-twentieth (100 µL) of equal-volume fractions
was separated by SDS-PAGE and transferred to nitrocellulose membranes.
A, Fractionation of AtRGP1 was determined by immunoblot analysis and
compared with that of the membrane marker BiP (ER). Because of the
presence of AtRGP1 and BiP in the same fractions, similar gradients
were analyzed in the presence of Mg+2 and showed that BiP
shifted to a denser part of the gradient, but AtRGP1 did not (data not
shown). B, AtRGP1 fractionation was also compared with that of other
known membrane markers, AtELP, AtPEP12p, and R28.
). Preliminary results of electron microscopy partially localize
AtELP to the trans-Golgi (S.U. Ahmed and N.V. Raikhel,
unpublished data). Our results support the Golgi localization of RGP
(Dhugga et al., 1997).
) in a manner that was reversed by using UDP-Glc, UDP-Xyl, and
UDP-Gal. Similar experiments revealed a single labeled protein when
total Arabidopsis soluble protein was analyzed (Fig.
6A). Reactions using total pea membrane
protein yielded the expected PsRGP1 doublet (Fig. 6A). Only pea seemed to have a clear doublet, since tobacco and maize showed only a single
protein labeled with UDP-Glc (Fig. 6A), and total protein from
Synechocystis sp. strain PCC6803 or purified GST protein showed no labeling (Fig. 6A).
View larger version (51K):
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Figure 6.
AtRGP1 is reversibly autoglycosylated. A, Total
protein (100-250 µg) from Arabidopsis (lane 1), pea (lane 2),
tobacco (lane 3), maize (lane 4), and cyanobacteria (lane 5); 5 µg of
purified GST (lane 6) and GST-AtRGP1p (lane 7) and 50 µg of the
Arabidopsis Suc-gradient fraction 10 (lane 8) were incubated with 0.1 µCi of UDP-[14C]Glc before analysis by SDS-PAGE and
autoradiography. Molecular mass is indicated in kilodaltons. B,
Displacement of bound radiolabel from GST-AtRGP1. Two micrograms of
GST-AtRGP1 was incubated with UDP-[3H]Glc for 10 min.
Various substrates were then added to a final concentration of 3 mm and the incubations were continued for 10 min more. Lane
1, No substrate added; lane 2, UDP; lane 3, Glc; lane 4, UDP-Glc, lane
5, UDP-Xyl; lane 6, UDP-Gal; and lane 7, UDP-Man. After the
second incubation, reactions were stopped and analyzed by SDS-PAGE
and autoradiography.
). AtRGP1 seems to behave in a similar way, since all of the
samples shown in Figure 6 were boiled in loading buffer containing 3%
SDS prior to analysis. To test further the nature of this association
in Arabidopsis, a purified GST fusion of AtRGP1 was prepared. The 68-kD
GST fusion, GST-AtRGP1p, was incubated with
UDP-d-[14C]Glc or
UDP-d-[3H]Glc in the absence of
metal ions and was shown to be glycosylated by UDP-Glc (Fig. 6). The
reaction with UDPd-[3H]Glc was
tested against UDP, Glc, UDP-Glc, UDP-Xyl, UDP-Gal, and UDP-Man. UDP,
UDP-Glc, UDP-Xyl, and UDP-Gal were able to displace the label from
GST-AtRGP1 (Fig. 6B), whereas Glc and UDP-Man were not (Fig. 6B). These
results demonstrate that AtRGP1, as shown for PsRGP1 (Dhugga et al.,
1997), is autoglycosylated independently of secondary factors.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) and monocots (Singh et al., 1995) but not in nonplant
systems suggests a plant-specific function.
), in which it has been suggested that
PsRGP1 is a single-copy gene. Elucidating the functional
role of AtRGP1-related Arabidopsis proteins such as AtRGP2 will be
pursued because they share a high degree of identity and yet include
subtle differences, e.g. a reduction of sequence identity at the N
terminus, which leads to the absence of a putative
N-myristoylation site in AtRGP2 (not shown).
) is a strong
indication that these motifs are conserved in different polysaccharide-synthesizing enzymes. Sequence comparisons with previously defined UDP-Glc-binding sites (Delmer and Amor, 1995; Pear
et al., 1996; Saxena and Brown, 1997) showed that AtRGP1 contains a
similar motif that may be involved in its binding to UDP-sugars. This
motif shares 40% sequence identity with U1, the cotton CelA
UDP-Glc-binding domain.
isolated a sweet corn protein based on its
glycosylation with UDP-Glc, which they named amylogenin. The protein
was digested and the sequence from eight tryptic peptides accounted for
about 40%. Dhugga et al. (1997) suggested that this protein be named
ZmRGP1, since the tryptic peptide sequences were nearly identical to
PsRGP1 and AtRGP1. A single amino acid, Arg-158, was found to be
labeled with UDP-d-[14C]Glc, in
accordance with the single glycosylation of PsRGP1 (Dhugga et al.,
1991). The U1 motif (Pear et al., 1996), a UDP-Glc-binding domain
predicted through its sequence conservation with the catalytic subunits
of enzymes involved in the polymerization of -glucosyl residues, as
well as UDP-Glc-binding proteins in other systems, may function in
UDP-Glc binding in AtRGP1 prior to the glycosylation of this protein at
Arg-158.
) and possibly in maize (Epel et al., 1996). All of the membrane
markers used in this study, with the exception of ARA4, have been
extensively characterized in our laboratory (Ahmed et al., 1997;
Bar-Peled and Raikhel, 1997; Conceição et al., 1997). We
were able to show that AtRGP1 is found in membrane-containing fractions
other than that of the ER or the plasma membrane, presumably the Golgi
apparatus. The accumulation of ARA4 (Ueda et al., 1996) in the soluble
fraction (Fig. 4) is typical of small GTP-binding proteins, which exist
in both cytosolic and membrane-associated forms. Sequence analysis of
AtRGP1 suggests that it is a soluble protein. Membrane-association
experiments show that most of AtRGP1 is indeed soluble, but that a
small fraction can be found peripherally associated with membranes.
Such a distribution within the cell suggests that the reaction of
AtRGP1 with UDP-sugars takes place in the cytoplasm.
). AtRGP1's reversible glycosylation, its prominent residence
in the cytoplasm, where nucleotide sugars are found, and its transient
association with membranes suggests that it functions as a carrier of
UDP-sugars from the cytoplasm to membranes such as the Golgi apparatus.
), suggest that AtRGP1 may play
a role in cell wall biosynthesis.
1
This research was supported by a grant from the
U.S. Department of Energy (no. De-FG02-91ER20021).
FOOTNOTES
*
Corresponding author; e-mail nraikhel{at}pilot.msu.edu; fax
1-517-353-9168.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
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J Biol Chem
266:
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[CrossRef][ISI][Medline]
