| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol, September 2001, Vol. 127, pp. 360-371
The Catalytic Site of the Pectin Biosynthetic Enzyme
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
-1,4-Galacturonosyltransferase (GalAT) is an enzyme required for
the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes
co-localized in linear and discontinuous sucrose gradients with latent
UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase
activities, enzyme markers for the endoplasmic reticulum and the
mitochondria, respectively. GalAT and latent UDPase activities were
separated from the majority (80%) of callose synthase activity, a
marker for the plasma membrane, suggesting that little or no GalAT is
present in the plasma membrane. GalAT activities in proteinase
K-treated and untreated Golgi vesicles were similar, whereas no GalAT
activity was detected after treating Golgi vesicles with proteinase K
in the presence of Triton X-100. These results demonstrate that the
catalytic site of GalAT resides within the lumen of the Golgi. The
products generated by Golgi-localized GalAT were converted by
endopolygalacturonase treatment to mono- and di-galacturonic acid,
thereby showing that GalAT synthesizes 1
4-linked
-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is
present in the Golgi apparatus. Together with prior results of in vivo
labeling and immunocytochemical studies, these results show that pectin
biosynthesis occurs in the Golgi. A model for the biosynthesis of the
pectic polysaccharide HGA is proposed.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
Pectins are a family of
polysaccharides present in all plant primary walls (O'Neill et al.,
1990
). Homogalacturonan (HGA) accounts for approximately 60% of the
total pectin in plants (O'Neill et al., 1990; Mohnen et al., 1996).
HGA is a linear polymer composed of 14-linked
-D-galactosyluronic acid residues (GalA) that are often
methyl esterified at the C-6 carboxyl group (Mort et al., 1993) and may also be O-acetylated at O-2 and
O-3 (Ishii, 1997). The GalA residues may, in some plants, be
substituted at O-3 with a 
-linked xylosyl residue (Schols
et al., 1995; Yu and Mort, 1996).
An enzyme that catalyzes the transfer of GalA from UDP-GalA onto
endogenous HGA was first identified in particulate suspensions from
Phaseolus aureus and was named polygalacturonate
4--galacturonosyltransferase (EC 2.4.1.43; Villemez et
al., 1965, 1966). A similar enzyme was characterized in
microsomal membranes from suspension-cultured tobacco (Nicotiana
tabacum L. cv Samsun) cells (Doong et al., 1995)
and solubilized from tobacco membranes (Doong and Mohnen, 1998). The
solubilized enzyme was shown to catalyze the addition of a single
-GalA residue onto O-4 of the nonreducing end
(Scheller et al., 1999) of exogenous oligomeric HGA acceptors (Doong
and Mohnen, 1998) and thus was named homogalacturonan
4--galacturonosyltransferase (HGA-GalAT).
No direct evidence has yet been reported for the subcellular
localization of the glycosyltransferases responsible for pectin biosynthesis. However, the results of in vivo labeling and
immunocytochemical analyses have suggested that pectin is synthesized
in the Golgi apparatus (Northcote and Pickett-Heaps, 1966; Harris and
Northcote, 1971; Staehelin and Moore, 1995). Immunocytochemical studies
using antibodies that recognize the pectic polysaccharides HGA and
rhamnogalacturonan I suggest that these pectic polysaccharides are
synthesized within different compartments of the Golgi apparatus (Zhang
and Staehelin, 1992) and are transported from the Golgi in vesicles
that migrate to, and fuse with, the plasma membrane. The
polysaccharides are then released into the extracellular space and
incorporated into the wall (Northcote and Pickett-Heaps, 1966).
We now provide evidence that an -1,4-galacturonosyltransferase
(GalAT) from pea (Pisum sativum L. var. Alaska) epicotyls, with properties similar to the previously described HGA-GalAT from
tobacco, is located in the Golgi apparatus and that the catalytic site
of this enzyme is in the Golgi lumen. The third internodes of etiolated
pea epicotyls were used for this study since the different subcellular
membranes from this tissue can be separated using Suc density
gradients, making such tissue useful for topological and localization
studies of enzymes involved in cell wall polysaccharide biosynthesis
(Brummell et al., 1990; Orellana et al., 1997; Wulff et al., 2000). Our
data provide strong evidence that HGA is synthesized in the Golgi
apparatus and a model for the biosynthesis of this pectic
polysaccharide is proposed.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
Subcellular Localization of GalAT Activity in Pea Membranes
Subcellular membrane fractions obtained by linear Suc gradient
centrifugation of homogenates from etiolated pea third internodes were
analyzed for GalAT activity and for organelle-specific enzyme activity
(Fig. 1). GalAT activity was detected
almost exclusively in fractions 26 through 30 (1.17-1.14 g
cm
3; Fig. 1a). The presence of GalAT activity
in such a limited number of fractions suggests that it is associated
with an organelle of a distinct density. Enzyme marker assays for the
ER and for the mitochondria (antimycin A-insensitive NADH:Cyt
c reductase and Cyt c oxidase, respectively)
showed that the ER (fractions 30-40, 1.14-1.10 g
cm3) and mitochondria (fractions 19-22,
1.20-1.18 g cm3) were present in a different
subset of fractions (Fig. 1b) than those that contained the greatest
amount of GalAT activity.
|
Latent UDPase activity is a well-characterized Golgi-specific
enzyme marker (Orellana et al., 1997) that is detected
following the permeabilization of membranes with detergent. The
greatest amount of latent UDPase activity was present in fractions 24 through 32 of a density of 1.17 to 1.14 g
cm3 (Fig. 1c). These fractions coincide with
the fractions that contain the greatest amount of GalAT activity,
suggesting that GalAT is a Golgi-localized enzyme.
To confirm that GalAT is present in a Golgi-enriched fraction,
pea homogenates were separated using discontinuous Suc gradient centrifugation. Each fraction (Fig. 2)
was analyzed for GalAT activity and for membrane marker activities
(Table I). Latent UDPase activity was
highest in the membranes that banded at the 8% to 33% (w/w) Suc
interface suggesting that these membranes were Golgi enriched (Tanford
and Reynolds, 1976). The Golgi-enriched fraction also contained
approximately 90% of the total GalAT activity (Table I). Some (21% of
total) ER-specific enzyme marker activity was also detected in the
Golgi-enriched fraction. However, the ER-enriched fraction (33%-38%
[w/w] Suc interface) contained no GalAT activity (Table I).
These results, when taken together with the results shown in Figure 1,
strongly suggest that the GalAT from pea membranes is a Golgi-localized
enzyme.
|
|
The biosynthesis of cellulose and callose has been shown to occur at
the plasma membrane (Frederikson and Larsson, 1989; Delmer, 1999), and
the possibility could not be discounted that GalAT may also be
localized to the plasma membrane. Thus, experiments were conducted,
using discontinuous Suc gradient centrifugation, to separate Golgi
membranes from plasma membranes. The gradient fractions were assayed
for GalAT activity, latent UDPase, and callose synthase activity, a
known marker for plasma membrane vesicles in plants (Morre et
al., 1987; Dhugga and Ray, 1994; Table
II). Callose synthase activity was
present in all of the discontinuous Suc gradient fractions assayed.
These results indicate that the Golgi-enriched membranes obtained using
discontinuous Suc gradients also contained some plasma membranes. Thus,
the possibility cannot be ruled out that GalAT is localized to a subset of plasma membrane vesicles that co-localize with Golgi membranes. Nevertheless, because approximately 80% of the callose synthase activity is located in fractions that are distinct from those that
contain the majority of the GalAT and latent UDPase activities (Table
II), these results most strongly support the conclusion that GalAT is
Golgi localized and is not a component of the plasma membrane.
|
Characterization of the Products Made by GalAT in Enriched Golgi Membranes from Pea
The subcellular fractionation of GalAT activity with the Golgi-enriched fraction from pea suggested that a pectin biosynthetic enzyme was localized in the Golgi. To test whether HGA is formed by the GalAT in pea Golgi, the [14C]-labeled products generated by incubating UDP-[14C]GalA with intact pea Golgi were analyzed by size exclusion chromatography (SEC) and by high performance anion-exchange chromatography (HPAEC). The products were analyzed both before and after treatment with base (to remove methyl and other esters), with endopolygalacturonase (EPGase; to fragment HGA), and with base plus EPGase. Fractions were collected, analyzed by scintillation counting, and the elution times of the radioactive products compared with the elution times of GalA, oligogalacturonides (OGAs), and dextran molecular mass standards.
The SEC elution profiles of untreated and the EPGase-treated products
from intact pea Golgi are shown in Figure
3. The untreated product gave a peak
(20% of the total radioactivity) with a retention time earlier than
the 500-kD dextran standard, and a peak (42% of the total
radioactivity) that co-eluted with GalA. Some radioactivity eluted
between these two peaks and most likely corresponds to variously sized
HGA. EPGase treatment of the intact product resulted in the conversion
of the high-molecular mass material into products that have the
same retention time as mono- and diGalA (Fig. 3). These results suggest
that most of the [14C]GalA-labeled product made
using intact Golgi membranes is -1,4-linked HGA.
|
The untreated and EPGase-treated products, and the products that were formed upon base and base plus EPGase treatment, were analyzed by HPEAC (Fig. 4, a-d). The untreated product contained a peak (27% of total radioactivity) that had a retention time similar to GalA, as well as a series of smaller peaks (32% of total radioactivity) that eluted at retention times comparable with medium-sized OGAs (DPs of 12 and 15-20; Fig. 4a). Base treatment of the untreated product did not alter the retention time of the radioactive peaks, suggesting that the products formed by intact pea Golgi were not extensively methoxylated or otherwise esterified (Fig. 4c). EPGase treatment of the untreated (Fig. 4b) or base-treated (Fig. 4d) products resulted in the appearance of diGalA, an increase in the amount of GalA, and the disappearance of the mid-sized OGAs, thereby confirming that the oligomeric peaks in the untreated and the base-treated products were HGA.
|
The amount of radiolabeled mono- and diGalA recovered after EPGase digestion and HPAEC was not equal to the amount of radioactivity detected as the series of small peaks in the untreated product (compare Fig. 4, a with b). Furthermore, the absence of a peak corresponding to the high-molecular mass product (see Fig. 3) in either Figure 4, a or c, suggests either that the high-molecular mass material was unable to elute as a defined peak on the anion-exchange column or that the material was unable to pass through the 0.2-µm filters used prior to HPAEC analysis (see "Materials and Methods"). Analysis of the 0.2-µm filters revealed that approximately 47% and 26% (for untreated and base-treated products, respectively) of the total label incorporated into product remained on the filter following filtration.
In a preliminary study, we found that the amount of radiolabeled product formed by membrane-bound GalAT from pea increased with the addition of exogenous OGA acceptors in the presence of 0.1% (v/v) Triton X-100 (data not shown). Therefore, the product formed by Golgi membranes in the presence of 0.1% (v/v) Triton X-100 and an OGA acceptor (DP 14) was analyzed to confirm that the GalAT produced HGA under these conditions.
Several differences were observed between the products made by intact and detergent-permeabilized Golgi membranes. For example, approximately 47% of untreated product made by intact Golgi bound to the HPLC prefilter, whereas only 12% of the untreated product from Triton X-100-permeabilized Golgi bound to the filter. Comparable amounts of radioactive product (83% for intact Golgi and 98% for permeabilized Golgi) passed through the filter after base and EPGase treatment. These results suggested that the products formed using detergent-permeabilized Golgi and exogenous oligomeric HGA acceptors were smaller than, and/or had different characteristics than, the products made by intact Golgi vesicles.
The [14C]-labeled products formed using 0.1%
(v/v) Triton X-100-permeabilized pea Golgi in the presence of an
OGA acceptor (DP 14) were analyzed by SEC and HPAEC (Figs.
5 and 6)
before and after base treatment, EPGase treatment, and base plus EPGase
treatment. SEC of the untreated product (Fig. 5) gave two peaks that
eluted with the same retention time as an authentic OGA of DP 15 (15 mer, 32% of total) and GalA (22% of total), respectively. The peak
that eluted before GalA was shown (see below) to be GalA-1-phosphate (GalA1-P; 44% of total; Fig. 5). The radioactive 15-mer peak was completely converted by EPGase treatment into peaks that had the same
retention time as GalA and diGalA (Fig. 5). No large molecular mass
peak was detected in the untreated product formed by Triton X-100-treated pea Golgi, suggesting that in the presence of exogenous acceptor the GalAT in Triton X-permeabilized membranes does not synthesize a large molecular mass product. Similar results have been
obtained with a GalAT solubilized from tobacco membranes (Doong and
Mohnen, 1998).
|
|
HPEAC analysis of the products made by Triton X-100-treated pea Golgi in the presence of an OGA acceptor are consistent with the results obtained by SEC. The untreated product (Fig. 6a) eluted as three predominant peaks of radioactivity that co-eluted with GalA, GalA-1-P (elutes slightly earlier than tetraGalA, see below), and the OGA 15 mer, respectively. A small amount of larger sized material was also present.
Two methods were used to establish that the radiolabeled peak that elutes between tri-GalA and tetra-GalA is GalA-1-P. Authentic GalA-1-P was chromatographed by HPAEC, fractions collected, and the uronic acid content of each fraction determined colorimetrically. The retention time of the radiolabeled peak and GalA-1-P were the same (data not shown). To confirm that the putative radiolabeled GalA-1-P was GalA-1-P, radiolabeled product generated by permeabilized Golgi in the presence of OGA of DP 12 was recovered by extraction of the product with chloroform:methanol and a portion of the aqueous phase was treated with alkaline phosphatase. The untreated (Fig. 7a) and phosphatase-treated products (Fig. 7b) were separated by HPAEC. As anticipated, the putative GalA-1-P peak disappeared after treatment with phosphatase and the amount of GalA increased. Phosphatase treatment did not change the retention time of OGA (DP 13) or GalA. These results establish that the radioactive product that elutes by HPEAC between tri-GalA and tetra-GalA is phosphorylated GalA.
|
Base treatment of the intact product made by detergent-permeabilized Golgi did not shift the retention time of the peaks (Fig. 6c), suggesting that the products were not methoxylated or otherwise esterified. Treatment of the untreated or base-treated products with an EPGase resulted in the disappearance of the OGA 15 mer and larger peaks, the appearance of diGalA, and an increase in the amount of GalA (Figs. 6, b and d). These results show that most of the [14C]GalA-labeled product made in Triton X-100-permeabilized Golgi membranes in the presence of exogenous OGA 14 mer is OGA of DP 15.
The GalAT in permeabilized Golgi vesicles and the GalAT solubilized
from tobacco synthesize similar products. Therefore, it was possible
that 0.1% (v/v) Triton X-100 was solubilizing the enzyme from
Golgi membranes rather than permeabilizing the membranes. To test this,
Golgi vesicles were treated with 0.1% (v/v) Triton X-100,
centrifuged at 100,000g for 1 h, and the GalAT activity in the supernatant and in the pellet was determined (data not shown).
All of the activity was present in the pellet, suggesting that 0.1%
(v/v) Triton X-100 alone was not sufficient to solubilize GalAT
activity from Golgi membranes and that the changes in product formation
upon Triton X-100 treatment were due either to the permeabilization of
the Golgi membranes or to the effect of the detergent on the GalAT
itself. Nevertheless, the characteristics of the products formed by
GalAT from pea (Figs. 3-6) are consistent with the characteristics of
the membrane bound (Doong et al., 1995) and solubilized
forms of GalAT from tobacco (Doong and Mohnen, 1998).
Topography of GalAT in Pea Golgi
Pectin biosynthesis is believed to occur in the lumen of the Golgi
endomembrane system (Northcote and Pickett-Heaps, 1966; Zhang and
Staehelin, 1992) and it is likely that the glycosyltransferases, or at
least their catalytic sites, face the lumen of the Golgi. To test this
hypothesis, isolated Golgi membranes were treated with Proteinase K (a
protease that cannot pass through intact membranes) in the presence and
in the absence of 0.1% (v/v) Triton X-100 and then assayed for
GalAT activity. GalAT activity was similar in intact Golgi vesicles,
Triton X-100-treated vesicles, and proteinase K-treated vesicles (Fig.
8). However, GalAT activity in Golgi
membranes treated with both Triton X-100 and proteinase K was reduced
to levels comparable with that of boiled Golgi vesicles. These results
demonstrate that the intact Golgi membranes protect the catalytic site
of GalAT from Proteinase K digestion and strongly suggest that the
catalytic domain of GalAT is located within the lumen of the
Golgi.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
The results presented here provide the first direct enzymatic
evidence that GalAT is located in the Golgi apparatus. GalAT activity
localizes exclusively to fractions that contain the majority of the
latent UDPase activity, a well-characterized Golgi-specific enzyme
marker. A combination of detergent and proteinase K treatments show
that pea GalAT is a membrane-bound protein whose catalytic site faces
the lumen of the Golgi apparatus. The catalytic site of tobacco HGA
methyltransferase has also been localized to the lumen of the Golgi
apparatus (Goubet and Mohnen, 1999). Taken together, our results show
that pectin biosynthetic enzymes are present in the Golgi and confirm
that pectin biosynthesis occurs in the Golgi endomembrane system
(Northcote and Pickett-Heaps, 1966; Harris and Northcote, 1971; Zhang
and Staehelin, 1992).
A model summarizing our current understanding (Doong et al.,
1995; Liljebjelke et al., 1995; Doong and Mohnen,
1998) of the biosynthesis of HGA is shown in Figure
9. UDP-GalA, the substrate for GalAT, is
believed to be formed on the cytosolic side of the Golgi by the
epimerization of UDP-GlcA catalyzed by a UDP-glucuronate-4-epimerase (EC 5.1.3.6; Feingold et al., 1960; Feingold and Avigad,
1980; Mohnen, 1999) that is associated with Golgi membranes (K. Adams and D. Mohnen, unpublished data). The UDP-GalA is thought to be transported into the lumen of the Golgi apparatus by a proposed membrane-bound UDP-GalA:UMP antiport transport protein. Similar nucleotide sugar transporters involved in protein glycosylation have
been identified, purified, and cloned from several mammalian and yeast
sources (Hirschberg et al., 1998). GalAT, a Golgi-resident protein with
its catalytic site in the lumen of the Golgi, binds UDP-GalA and
catalyzes the transfer of GalA onto a growing HGA chain and the
production of UDP. The UDP is hydrolyzed in the lumen into inorganic
phosphate and UMP by a Golgi-resident membrane-bound nucleoside
diphosphatase (NDPase/UDPase) (Orellana et al., 1997). The UMP is
exported out of the Golgi in exchange for UDP-GalA by the proposed
UDP-GalA:UMP antiport transporter.
|
The products synthesized by the GalAT from pea are comparable
with the products synthesized by the GalAT from tobacco (Doong et
al., 1995; Doong and Mohnen, 1998). The GalAT in
detergent-permeabilized pea Golgi membranes produces a product
that is predominantly only one GalA residue longer than the exogenous
acceptor. This result is similar to that obtained with the solubilized
GalAT from tobacco, where the product generated is also only one
residue longer than the exogenous acceptor (Doong and Mohnen, 1998). In
contrast, the GalAT in intact pea Golgi and in tobacco membranes both
use endogenous acceptors to generate high-molecular- mass
products, with the product in pea (>500 kD) being larger than that
produced in tobacco (approximately 105 kD; Doong et al.,
1995). The number of GalA residues added onto the endogenous
acceptor(s) in the pea and tobacco membranes in vitro has not yet been determined.
There are a number of possible reasons why the pea
detergent-permeabilized and the tobacco-solubilized GalATs add only a
single GalA to the end of the exogenous acceptors. For example,
treating pea Golgi vesicles with detergent and exogenous OGA acceptors may cause the GalAT to preferentially use the exogenous OGAs as acceptors, whereas the GalAT in intact Golgi uses an endogenous acceptor. The exogenous OGA (e.g. DP 14) acceptor may not be the optimal substrate for the GalAT, whereas the endogenous product found
in intact Golgi membranes presumably contains the necessary structural
information for the enzyme to synthesize a polymeric product. Such
acceptor-mediated control of enzyme processivity has been demonstrated
for a mammalian -2,8-polysialic acid synthase (Kojima et
al., 1996), where the placement of an -1,6-linked Fuc
residue in the acceptor was shown to be necessary for polysialylation. It is also possible that treating Golgi membranes with Triton X-100
disrupts an enzyme complex (protein-protein or other interaction) or
removes an element that is required for polymer synthase activity. Protein-protein complexes involved in glycan biosynthesis have been
demonstrated for glycogenin, a self-glucosylating protein that
initiates glycogen biosynthesis (Lin et al., 1999).
The presence of GalA among the products formed by both intact and
detergent-permeabilized pea Golgi membranes, and the presence of
GalA1-P in the products formed by detergent-permeabilized pea Golgi
membranes, are a major difference between the reaction products formed
by pea and tobacco membranes (Doong and Mohnen, 1998). The presence of
GalA and GalA1-P most likely results from the hydrolysis of
UDP-[14C]GalA by a phosphodiesterase (Feingold
et al., 1958) and a phosphatase (Neufeld et al.,
1961). Such hydrolases have been reported to be released during the
homogenization and isolation of membranes from some plant tissues
(Villemez et al., 1966; Rodgers and Bolwell, 1992; Brickell
and Reid, 1996) and it is likely that such enzymes were released during
the preparation of the pea Golgi.
Several enzymes including latent UDPase (Orellana et al.,
1997) and mammalian glycosyltransferases (Paulson and Colley, 1989) such as the polysialyltransferases that function in the polysialylation of neural cell adhesion molecule and other N-linked
glycoproteins (for review, see Tsuji, 1996) have their catalytic sites
located in the lumen of the Golgi. Most Golgi-localized
glycosyltransferases are type II integral membrane proteins with a
single N-terminal membrane-spanning region and a lumenal C-terminal
catalytic domain (Paulson and Colley, 1989; Tsuji, 1996). On the other
hand, plasma membrane-localized polymer synthases such as hyaluronan
synthase (Weigel et al., 1997) and the putative cellulose synthase
(Delmer, 1999) are predicted to have multiple transmembrane-spanning
regions at the amino and carboxy termini and a large central domain
containing the catalytic site. The purification of the GalAT, the
determination of its amino acid sequence, and the identification of its
gene will be required to elucidate the structure of GalAT and will allow the comparison of GalAT structure with that of known
glycosyltransferases and polymer synthases.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
Chemicals
Uridine
diphosphate--D-[14C]GlcA
(UDP-[14C]GlcA, 293.6 mCi mmol1)
and uridine diphosphate--D-[14C]Glc
(UDP-[14C]Glc, 286.2 mCi mmol1)
were purchased from DuPont-New England Nuclear (Boston).
Uridine diphosphate--D-[14C]GalA
(UDP-[14C]GalA) was prepared by the epimerization of
UDP-[14C]GlcA according to Liljebjelke et al. (1995).
OGAs with a DP of 14 and a mixture of OGAs of DP 7 to 23 were prepared
according to Doong et al. (1995). Proteinase K was purchased from
Boehringer Mannheim (Indianapolis). Homogeneous EPGase (748 units
mL1, 1 unit releases 1 µmol GalA per min) from
Fusarium moniliforme was a gift from Carl Bergmann
(Complex Carbohydrate Research Center, Athens, GA). All other
reagents were purchased from Sigma (St. Louis).
Plant Materials
Pea (Pisum sativum L. var. Alaska) seeds were purchased from the Green Seed Company (Athens, GA). Seedlings were grown in moist vermiculite for 7 to 8 d at 25°C in the dark.
Linear Suc Gradient Fractionation of Pea Homogenates
Third internodes (from the cotyledon, 1 cm in length, 5 g
total weight) from 7- to 8-d-old etiolated peas (Pisum
sativum L. var Alaska) were excised and homogenized in 50 mM HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]/KOH, pH 7.0, containing 0.4 M Suc, 1 mM MgCl2,
10 mM KCl, 1 mM EDTA, and 1 mM
dithiothreitol using a razor blade and a mortar and pestle. The
homogenate was filtered through two layers of Miracloth (Calbiochem,
San Diego) and the filtrate centrifuged for 10 min at
1,000g. The 1,000g supernatant (5 mL) was
overlaid onto 24 mL of a 20% to 50% Suc (w/w) gradient (Munoz et
al., 1996) in 50 mM HEPES/KOH, pH 7.0 containing 0.1 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol and centrifuged for 3.5 h at
100,000g. Fractions (0.5 mL) were collected and analyzed
for marker enzyme activity. All manipulations were done at 4°C. Suc
concentrations were determined using an Abbe refractometer (model 3L,
Milton Roy Company, Rochester, NY) and the densities were calculated using an ambient temperature of 20°C.
Isolation of Golgi Membranes
Enriched Golgi membranes were isolated using discontinuous Suc
gradients according to the method of Munoz et al. (1996). All manipulations were done on ice or at 4°C. The third internodes (1 cm,
40-70 g total mass) of 7- to 8-d-old etiolated pea stem epicotyls were
excised and homogenized in 1 volume of buffer A (0.1 M
KH2PO, pH 6.65, containing 5 mM
MgCl2, 1 mM dithioerythritol, and 16% [w/w]
Suc) using a razor blade and a mortar and pestle. The homogenate was
filtered through two layers of Miracloth and centrifuged for 2 min at
1,000g. The supernatant was layered onto 38% (w/w) Suc
in buffer A (8 mL) and centrifuged for 90 min at 100,000g. The upper phase was removed without disturbing
the interface layer and replaced with 33% (w/w) Suc (15 mL) and 8%
(w/w) Suc (5 mL) in buffer A. The gradient was centrifuged for a
further 100 min at 100,000g. The pellet was resuspended
in 10 mM Tris, pH 7.5, containing 0.25 M Suc
(ST buffer) and stored at 
80°C. The membranes that
equilibrated at each of the interfaces were collected, diluted with an
equal volume of water, and centrifuged for 50 min at
100,000g. These pellets were resuspended in ST
buffer and stored at 80°C. In some experiments (Table II),
membranes from the 33% and 38% (w/w) Suc phases were also
collected and stored as described above. Otherwise, the 33% and 38%
Suc phase with no prior membrane collection was used for enzyme
analysis (Table I).
Enzyme Assays
Cyt c oxidase activity was measured as described
by Briskin et al. (1987) with some modifications. Cyt c
oxidase was assayed spectrophotometrically at 550 nm using a scanning
spectrophotometer (model UV-2101; Shimadzu, Columbus, MD). A portion
(35 µL) of each gradient fraction was added to 30 mM
K2HPO4, pH 7.4 (735 µL), containing 1 mM EDTA, 1 mM NaHCO3, 0.00045%
(v/v) Triton X-100, and 50 µM Cyt c
(reduced with sodium dithionite). The total activity of Cyt
c oxidase was calculated for each fraction using the
initial linear rate of Cyt c oxidation (decrease in
A550) and the extinction coefficient of Cyt
c (18.5 mM1
cm1).
Antimycin A-resistant NADH:Cyt c reductase was measured
according to a modification of Vannier et al. (1992). A portion (0.1 mL) of each gradient fraction was combined with 50 mM
KH2PO4, pH 7.5 (0.9 mL), containing 50 µM Cyt c, 0.3 mM NADH, 5 mM KCN, and 1 µM antimycin A. The linear
increase in A550 was taken as the rate of
Cyt c reduction and was used to calculate the total activity in each fraction.
Latent UDPase activity was measured according to Briskin et al. (1987)
with some modifications. A reaction mixture (15 µL) containing 6 mM UDP, 6 mM MgSO4, and 60 mM Tris/MES
[2-(N-morpholino)-ethanesulfonic acid], pH 6.5, with
or without 0.1% (v/v) Triton X-100, was added to 15 µL of
gradient fraction in a microtiter plate (Nunc, Suwanee, GA) and kept
for 15 min at 25°C. The reaction was stopped by the addition of Ames
reagent (270 µL) and processed as in Ames (1966). The
A690 of the sample was measured using a
Tikertek Multiscan ELISA plate reader (model MCC/340 MKII, Flow
Laboratories, McLean, VA) and was converted to the amount of Pi
released from UDP (UDPase activity) using a standard curve of
KH2PO4. Latent UDPase activity was considered
to be present in those fractions in which an increase in
A690 was observed when fractions were
incubated in the presence 0.1% (v/v) Triton X-100. The latent
UDPase activity reported in Tables I and II and in Figure 1 was
calculated by subtracting the amount of Pi released from UDP by enzyme
in the absence of Triton X-100 from the amount of Pi released from UDP
by enzyme in the presence of Triton X-100.
Callose synthase activity was measured according to a method by Dhugga
and Ray (1994) with some modifications. Gradient fractions (10 µL of
one-tenth-diluted sample) were incubated for 5 min in 10 mM
MOPS [3-(N-morpholino)-propanesulfonic acid], pH 7.0, containing 1 mM UDP-Glc, 10 mM cellobiose, 2 mM CaCl2, 0.3 mM spermine, 0.02% (v/v) Triton X-100, and 0.32 µM
UDP-[14C]Glc. The reaction was stopped by the addition of
70% (v/v) ethanol (2 mL) and the insoluble products were
collected by filtration through a GF/A glass microfiber disc (Whatman,
Maidstone, UK). The discs were washed with 70% (v/v) ethanol
(15 mL), dried, and the amount of radioactivity on the discs measured
by scintillation counting.
GalAT activity was measured as described by Doong et al. (1995) with
some modifications. Samples (10 µL) were incubated for 2 min in 50 mM HEPES, pH 7.8, containing 100 µg of OGAs (DP of 7-23), 1.1 µM UDP-[14C]GalA (specific
activity 293.6 mCi mmol1), 0.25 mM
MnCl2, 25 mM KCl, and 0.1% (v/v) Triton
X-100 in a total volume of 30 µL (unless otherwise stated).
The reaction was stopped by the addition of 700 µL
chloroform:methanol (3:2, v/v) and centrifuged to collect the
precipitable products. The product was washed twice with 500 µL of
65% (v/v) ethanol and the amount of radioactivity quantified
by scintillation counting.
All enzyme assays were conducted at room temperature with the exception
of the GalAT assay, which was conducted at 30°C. Protein concentrations were determined by the protein assay (Bio-Rad, Hercules,
CA) using bovine serum albumin as a standard. Uronic acids were
analyzed colorimetrically using the meta-hydroxybiphenyl assay
(Blumenkrantz and Asboe-Hansen, 1973).
Product Characterization
The products generated by GalAT in pea Golgi vesicles were
analyzed according to Doong et al. (1995). The products were formed using either detergent-permeabilized Golgi in the presence of OGAs of
DP 14 (14 mer) as exogenous acceptor or using intact Golgi without
exogenous acceptor.
Four separate GalAT reactions containing 12 µL of Golgi vesicles (approximately 43 µg of protein) and 5 µM UDP-[14C]GalA in 50 mM HEPES, pH 7.8, 0.25 mM MnCl2, and 25 mM KCl were incubated with or without 0.1% (v/v) Triton X-100 and 15 µg of OGA 14 mer for 35 min at 30°C in a total volume of 60 µL. The products were recovered, washed as described above, resuspended in 0.5 mL of 50 mM NaOAc, pH 6.0, containing 5 mM EDTA, and pooled (total volume 2 mL). Individual pools were analyzed as follows by either HPAEC or SEC. For example, a 500-µL aliquot was analyzed directly by HPAEC. A second aliquot of 500 µL was treated for 8 h at 30°C with a homogeneous EPGase (2 units) and the products generated were analyzed by HPAEC. A third aliquot of the pooled products (1 mL) was adjusted to pH 12 by the addition of 1 N NaOH and kept at 4°C for 8 h. The solution was adjusted to pH 5.0 with 1 N HCl and a portion (500 µL) was analyzed by HPAEC. The remaining base-treated product (500 µL) was treated with EPGase for 8 h and analyzed by HPAEC.
Alkaline Phosphatase Treatment of GalA-1-P
Aqueous extracts from a GalAT reaction were prepared by incubating 12 µL of Golgi vesicles (approximately 43 µg of protein) for 60 min at 30°C in 50 mM HEPES, pH 7.8, containing 2.5 µM UDP-[14C]GalA, and 15 µg of OGAs of DP 12, 0.25 mM MnCl2, 25 mM KCl, and 0.1% (v/v) Triton X-100 in a total volume of 60 µL. The reaction was terminated by the addition of chloroform:methanol (5:1, v/v, 300 µL) and 240 µL 50 mM NaOAc, pH 6.0, containing 5 mM EDTA was added. The mixture was centrifuged at 13,000g for 2 min. The aqueous upper phase was mixed with 300 µL of chloroform:methanol (5:1, v/v), centrifuged, and removed. The organic phase from the first centrifugation was mixed with 50 mM NaOAc, pH 6.0 (300 µL), containing 5 mM EDTA, centrifuged, and the aqueous phase was combined with the previously obtained aqueous phase. An aliquot (80 µL) of the combined aqueous phases was adjusted to pH 8.5 with Tris (10 mM). Water or 10 units of alkaline phosphatase was added, and the mixture kept for 60 min at 37°C. The reaction was stopped by heating at 85°C for 10 min. The resulting mixture was filtered (0.2 µm) and chromatographed by HPAEC. Fractions (1 mL) were collected and the amount of radioactivity in each fraction was quantified by liquid scintillation counting.
SEC of the [14C]-Labeled Product Generated Using Intact and Permeabilized Golgi
The products made by GalAT in intact Golgi without exogenous
acceptor, and the products formed by detergent-permeabilized (0.1%
[v/v] Triton X-100) Golgi in the presence of exogenous OGA 14 mer (15 µg) were separated using a Superose12 HR10/30 SEC column (Pharmacia, Uppsala). The column was eluted with 50 mM
sodium acetate containing 5 mM EDTA at 0.35 mL
min1. Fractions (0.5 mL) were collected and the
radioactivity in a portion (400 µL) of each fraction was determined
by scintillation counting. The SEC elution profiles of dextran
molecular mass standards, GalA, triGalA, and OGAs of a DP of 7, 12, 15, and 16 were detected using a pulsed electrochemical detector (Dionex,
Sunnyvale, CA) with post-column addition of 400 mM NaOH at
a flow rate of 0.2 mL min1 (see below).
HPAEC of the [14C]-Labeled Product Made Using Intact and Permeabilized Golgi
[14C]GalA-labeled product generated by GalAT in
pea Golgi vesicles, and GalA and OGA standards, were filtered (0.2-µm
nylon microfilterfuge tubes, Rainin Instrument Co., Inc., Woburn, MA) and chromatographed on a CarboPac PA100 column (4 × 250 mm)
fitted with a CarboPac PA100 guard column (3 × 25 mm) using a DX
500 LC (Dionex, Sunnyvale, CA). The column was eluted at 1 mL
min1 with a gradient formed from ultra-pure deionized
water (eluant A) and 1 M sodium acetate containing 5 mM EDTA (eluant B) as follows: 0% (v/v) B at 0 to 2 min,
40% (v/v) B at 17 min, 80% (v/v) B at 49 min, 100% (v/v) B at 50 to
52 min, and 0% (v/v) B at 57 min (modified from Doong et
al., 1995). The non-radiolabeled standards were detected
by pulsed electrochemical detection after post-column addition of 400 mM NaOH at 0.5 mL min1. The pulse sequence
for the electrochemical detector was 0.05 V at 0 s, 0.05 V at
0.2 s (begin integration), 0.05 V at 0.4 s (end integration),
0.75 V at 0.41 to 0.6 s, and 0.15 V at 0.61 to 1.0 s.
Fractions (0.75 mL) were collected, an aliquot (500 µL) of each
fraction was combined with 10 volumes of scintillation cocktail
(Scintiverse, Fisher Scientific, Norcross, GA), and the amount of
radioactivity was measured using a scintillation counter. Quenching by
salt and/or base was found to be negligible.
Proteinase K Digestion
Proteinase K treatment of Golgi vesicles was conducted according
to Orellana et al. (1997). In brief, Golgi vesicles (100 µg protein)
were incubated for 30 min at 30°C in 10 mM Tris, pH 7.5, containing 0.25 M Suc and 1 mM
MgCl2 (100 µL total volume) in the presence and absence
of 0.1% (v/v) Triton X-100 and 4 µg Proteinase K. Proteolysis was stopped by the addition of 1 mM phenylmethylsulfonylfluoride and 10 µL of each treatment was brought to a final concentration of 0.1% (v/v) Triton X-100 and
assayed for GalAT activity in the presence of exogenous OGA acceptors.
| |
ACKNOWLEDGMENTS |
|---|
We thank Lorena Norambuena for her assistance with the Suc gradients and the marker enzyme assays and Malcolm O'Neill for critical reading of the manuscript.
| |
FOOTNOTES |
|---|
Received February 2, 2001; returned for revision April 2, 2001; accepted June 1, 2001.
1 This work was supported by the National Science Foundation (grant no. INT-9722509 to D.M.), by the National Research Initiative (competitive U.S. Department of Agriculture award no. 98-35304-6772 to D.M.), by Fondecyt (grant no. 1000675 to A.O.), by the Programa de Cooperacion Internacional from Conicyt, Chile (grant to A.O.), by a Department of Energy-funded center grant (no. DE-FG05-93-ER20097), and by the Eastman Chemical Company, Kingsport, TN (fellowship to J.D.S.).
* Corresponding author; e-mail dmohnen{at}ccrc.uga.edu; fax 706-542-4412.
| |
LITERATURE CITED |
|---|
|
TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
|
|---|
-D-galactan.
In
J Visser, AGJ Voragen, eds, Pectins and Pectinases. Elsevier Sciences, Amsterdam, pp 47-53-D-glucan synthase activity from pea tissue: two polypeptides of 55 kDa and 70 kDa copurify with enzyme activity.
Eur J Biochem
220: 943-953[Web of Science][Medline]-galacturonosyltransferase and its products from membrane preparations of tobacco (Nicotiana tabacum L. cv samsun) cell suspension cultures.
Plant Physiol
109: 141-152[Abstract]-glucan synthase by Ca2+, spermine, and cellobiose: localization of activator sites using inside out plasma membrane vesicles.
Physiol Plant
77: 196-201[CrossRef]1,6-linked fucose and a polypeptide chain for polysialylation.
J Biol Chem
271: 19457-19463This article has been cited by other articles:
|
|
 |
|
  S. Wolf, G. Mouille, and J. Pelloux Homogalacturonan Methyl-Esterification and Plant Development Mol Plant, September 1, 2009; 2(5): 851 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yang, L. Bar-Peled, L. Gebhart, S. G. Lee, and M. Bar-Peled Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase J. Biol. Chem., August 7, 2009; 284(32): 21526 - 21535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Arsovski, M. M. Villota, O. Rowland, R. Subramaniam, and T. L. Western MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana J. Exp. Bot., July 1, 2009; 60(9): 2601 - 2612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Mast, L. Donaldson, K. Torr, L. Phillips, H. Flint, M. West, T. J. Strabala, and A. Wagner Exploring the Ultrastructural Localization and Biosynthesis of {beta}(1,4)-Galactan in Pinus radiata Compression Wood Plant Physiology, June 1, 2009; 150(2): 573 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ibar and A. Orellana The Import of S-Adenosylmethionine into the Golgi Apparatus Is Required for the Methylation of Homogalacturonan Plant Physiology, October 1, 2007; 145(2): 504 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Egelund, B. L. Petersen, M. S. Motawia, I. Damager, A. Faik, C. E. Olsen, T. Ishii, H. Clausen, P. Ulvskov, and N. Geshi Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-{alpha}-D-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II PLANT CELL, October 1, 2006; 18(10): 2593 - 2607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bacic Breaking an impasse in pectin biosynthesis PNAS, April 11, 2006; 103(15): 5639 - 5640. [Full Text] [PDF]
|
|
|
|
|
|
J. D. Sterling, M. A. Atmodjo, S. E. Inwood, V. S. Kumar Kolli, H. F. Quigley, M. G. Hahn, and D. Mohnen From the Cover: Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase PNAS, March 28, 2006; 103(13): 5236 - 5241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bosch, A. Y. Cheung, and P. K. Hepler Pectin Methylesterase, a Regulator of Pollen Tube Growth Plant Physiology, July 1, 2005; 138(3): 1334 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Egelund, M. Skjot, N. Geshi, P. Ulvskov, and B. L. Petersen A Complementary Bioinformatics Approach to Identify Potential Plant Cell Wall Glycosyltransferase-Encoding Genes Plant Physiology, September 1, 2004; 136(1): 2609 - 2620. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Urbanowicz, C. Rayon, and N. C. Carpita Topology of the Maize Mixed Linkage (1->3),(1->4)-{beta}-D-Glucan Synthase at the Golgi Membrane Plant Physiology, February 1, 2004; 134(2): 758 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. N. Gorshkova, T. N. Erokhina, T. A. Stroganova, N. E. Yelina, A. A. Zamyatnin Jr, N. O. Kalinina, J. Schiemann, A. G. Solovyev, and S. Yu. Morozov Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata J. Gen. Virol., April 1, 2003; 84(4): 985 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bouton, E. Leboeuf, G. Mouille, M.-T. Leydecker, J. Talbotec, F. Granier, M. Lahaye, H. Hofte, and H.-N. Truong QUASIMODO1 Encodes a Putative Membrane-Bound Glycosyltransferase Required for Normal Pectin Synthesis and Cell Adhesion in Arabidopsis PLANT CELL, October 1, 2002; 14(10): 2577 - 2590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wu, F. Liang, B. Hong, J. C. Young, M. R. Sussman, J. F. Harper, and H. Sze An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress Plant Physiology, September 1, 2002; 130(1): 128 - 137. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
-1,4-Galacturonosyltransferase Is Located in the Lumen of the
Golgi
) and Suc density (
). The data
were obtained from a single analysis of each fraction. Similar results
were obtained in three separate gradients. b, Cytochrome (Cyt)
c oxidase (
) and antimycin A-resistant NADH Cyt
c reductase (
), enzyme markers for mitochondria and
endoplasmic reticulum (ER), respectively. Activities are expressed as
the percentage of the highest total activity (191.2 nmol Cyt
c reduced min
)
products recovered following incubation of intact Golgi membranes with
4.7 µM UDP-[14C]GalA in the
absence of exogenous acceptor were separated by SEC. The amount of
radioactivity in each fraction was determined by scintillation
counting. The cpm of each fraction minus the background is shown. Base
(0.1 N NaOH) was included in the eluant solutions to enable
electrochemical detection of GalA and dextran molecular mass standards
by pulsed electrochemical detection.
