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Plant Physiol. (1998) 117: 1007-1014
Metabolism of Uridine 5
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Uridine 5
-diphosphate-glucose
(UDP-Glc) is transported into the lumen of the Golgi cisternae, where
is used for polysaccharide biosynthesis. When Golgi vesicles were
incubated with UDP-[3H]Glc, [3H]Glc was
rapidly transferred to endogenous acceptors and UDP-Glc was
undetectable in Golgi vesicles. This result indicated that a
uridine-containing nucleotide was rapidly formed in the Golgi vesicles.
Since little is known about the fate of the nucleotide derived from
UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc
by incubating Golgi vesicles with [
-32P]UDP-Glc,
[
-32P]UDP-Glc, and [3H]UDP-Glc and
identifying the resulting products. After incubation of Golgi vesicles
with these radiolabeled substrates we could detect only uridine
5-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be
detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The
by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the
lumen, indicating that they were able to exit the Golgi lumen. The exit
of UMP was stimulated by UDP-Glc, suggesting the presence of a putative
UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi
was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.
Glycosyltransferases, key enzymes in the biosynthesis of
polysaccharides, utilize nucleotide sugars as substrates in this essential cellular process (Brett and Waldron, 1996 UDP and other nucleoside diphosphates have been described as inhibitors
of glycosyltransferases (Ray et al., 1969 Since the metabolism of the nucleotide moiety of nucleotide sugars in
the lumen of the Golgi cisternae may have an impact on the synthesis of
polysacccharides in this organelle, we decided to analyze the
metabolism of UDP-Glc in Golgi vesicles from the elongating region of
etiolated pea (Pisum sativum L.) stems, focusing on the
nucleotide moiety. The results in this study demonstrate that upon
incorporation of UDP-Glc into Golgi vesicles, Glc is quickly
transferred to endogenous acceptors, and UMP and Pi are formed in the
lumen of Golgi vesicles as end products with no further hydrolysis of
UMP. The results also show that UMP exits the vesicles by a mechanism
that is stimulated by the entry of UDP-Glc, whereas Pi leaves the
vesicles by a mechanism that is independent of the entry of UDP-Glc,
suggesting that a putative UDP-Glc/UMP antiporter and Pi transporter
could be localized in the Golgi membrane.
Materials
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). Different lines
of evidence suggest that polysaccharide biosynthesis takes place in the
lumen of the Golgi cisternae, and that transport of nucleotide sugars
into the lumen is required to make the substrate available to the
glycosyltransferases (Zhang and Staehelin, 1992; Muñoz et al.,
1996). Once in the lumen, the sugar is transferred to endogenous
acceptors, which can be polysaccharides at various stages of synthesis,
glycoproteins, or glycolipids. However, in cells from elongating
tissues, the activity of the Golgi apparatus is mainly dedicated to
pectin and hemicellulose synthesis (Driouch et al., 1993). The other
product of the transfer reaction is the nucleoside diphosphate, which
is usually UDP, but it can be GDP or CDP as well (Brett and Waldron,
1996).
; Fredrikson and Larsson,
1992) and therefore they need to be metabolized to avoid inhibition of
polysaccharide biosynthesis. A Golgi nucleoside diphosphatase is
located in the Golgi membrane with its active site facing the lumen of
Golgi cisternae. For many years its activity has been correlated with
polysaccharide biosynthesis, suggesting that it could play a role in
this process (Dauwalder et al., 1969; Ray et al., 1969; Mitsui et al.,
1994; Orellana et al., 1997). The products of the reaction catalyzed by
this enzyme are UMP and Pi. According to our current hypothesis of the
topology of reactions leading to polysaccharide biosynthesis in the
Golgi apparatus, these metabolites should be produced in the lumen of the Golgi cisternae and then return to the cytoplasm, otherwise they
would accumulate in this compartment, inhibiting the synthesis of
additional polysaccharides. However, we do not know whether UMP is
formed in the Golgi vesicles and then further metabolized to uridine
and Pi. Previous studies from our laboratory showed that the
incorporation of UDP-Glc into the lumen of Golgi vesicles was coupled
to the exit of a uridine-containing nucleotide (Muñoz et al.,
1996), suggesting an antiporter mechanism. In addition, these data also
demonstrate that the nucleotide moiety exits the Golgi cisternae,
however, the exact nature of this uridine-containing nucleotide was not
known.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-32P]ATP (3000 Ci/mmol),
[-32P]UTP (3000 Ci/mmol), and
[5,63H]UTP (37 Ci/mmol) were purchased from
DuPont-NEN. UDP-[3H]Glc (4.5 Ci/mmol) was
purchased from Amersham. Nonradioactive UDP, UDP-Glc, and buffer
reagents were purchased from Sigma. Suc was high purity and obtained
from ICN. Proteinase K was from Merck (Darmstadt, Germany).
PEI-cellulose TLC plates were purchased from Aldrich.
Preparation of a Golgi-Enriched Vesicle Fraction
Pea (Pisum sativum var Alaska) seedlings were grown in moist vermiculite for 7 to 8 d in the dark at 25°C. Stem segments (1 cm) were excised from the elongating region of the hypocotyls. Vesicles were obtained as described by Muñoz et al. (1996) and Orellana et al. (1997). Pea stems were homogenized using
razor blades in the presence of 1 volume of 0.5 M Suc, 0.1 M KH2PO4, pH
6.65, 5 mM MgCl2, and 1 mM DTT. The homogenate was filtered through Miracloth
(Calbiochem) and centrifuged at 1,000g for 5 min. The
supernatant was loaded onto a 1.3 M Suc cushion and
centrifuged at 100,000g for 70 min. The upper phase was
removed, leaving, and without disturbing, the interface fraction. On
top of this, a discontinuous gradient was formed by adding 1.1 and 0.25 M Suc cushions and centrifuged for 90 min at
100,000g. The interface 0.25/1.1 M Suc fraction
was collected, diluted in 1 volume of distilled water, and centrifuged
at 100,000g for 35 min. The pellet was resuspended, using a
Dounce homogenizer, in STM buffer containing 0.25 M Suc, 1 mM MgCl2, and 10 mM
Tris-HCl, pH 7.5 (Suc-Tris-Mg buffer). The vesicles were kept at
70°C until use. All of the procedures were performed on ice or at
4°C. The UDPase latency of the vesicles varied from different
preparations between 85 and 90%. The characterization of this fraction
using marker enzymes has been described previously (Muñoz et al.,
1996).
UDPase Activity Detected in Native Polyacrylamide Gels
Native PAGE and detection of UDPase activity was done as described by Bollag and Edelstein (1991). A 10% acrylamide gel containing the
samples was run for 4 h at 80 V. Afterward, the gel was incubated for 15 min at 37°C in a solution containing 0.1 M
Tris-Maleate, 1.5 mM
Pb(NO3)2, 10 mM
MgSO4, 5% Glc, and 3 mM UDP. Then
the gel was rinsed with several changes of water and the active band
was visualized with 1%
(NH4)2S.
Synthesis and Purification of [-32P]UDP-Glc,
[-32P]UDP-Glc, and [3H]UDP-Glc
-32P]UDP-Glc was done as
described by Orellana et al. (1997). This method consists of the
transfer of the -phosphate present in
[-32P]ATP to Glc-1-P in a series of
reactions catalyzed by hexokinase and phosphoglucomutase. Then the
phosphate group present in Glc-1-32P is
transferred to UDP-Glc through the reaction catalyzed by the UDP-Glc
pyrophosphorylase coupled to inorganic pyrophosphatase. Synthesis of
[-32P]UDP-Glc and
[3H]UDP-Glc was done by using
[-32P]UTP and
[3H]UTP, respectively, in the reaction
catalyzed by the UDP-Glc pyrophosphorylase coupled with the removal of
pyrophosphate by the inorganic pyrophosphatase, exactly as described by
Muñoz et al. (1996). The radioactive products were purified as
described by Dhugga and Ray (1994) and Muñoz et al. (1996) using
charcoal. Purity was checked by TLC using PEI-cellulose plates using 1 N acetic acid as the mobile phase for 3 cm, and then 1 N acetic acid-LiCl 3 M (90:10, v/v) for 15 cm.
Purity was also verified by HPLC, demonstrating that the radioactive
compounds exhibit the same retention time as standard UDP-Glc.
Metabolism of Nucleotide-Radiolabeled UDP-Glc
Golgi vesicles (100 µg of protein) were incubated with 1 µM [-32P]UDP-Glc,
[-32P]UDP-Glc, or
[3H]UDP-Glc in a medium containing 0.25 M Suc, 1 mM MgCl2, and 10 mM Tris-HCl, pH 7.5. After incubation the reaction was heat
inactivated by placing the samples in a boiling water bath for 1 min
and then on ice. The samples were analyzed by TLC using the same mobile system described above. When [3H]UDP-Glc was used,
a control was carried on using vesicles that were previously boiled for
3 min. After the TLC separation the plate was cut in 1-cm strips and
counted in a liquid-scintillation counter. When
[-32P]UDP-Glc or
[-32P]UDP-Glc was used, the TLC plate
was exposed to autoradiography with an enhancing screen at 70°C.
The migration of labeled compounds on TLC was compared with possible
products derived from UDP-Glc hydrolysis.
UDP-Glc Incorporation into Golgi Vesicles
Incorporation of [-32P]UDP-Glc,
[-32P]UDP-Glc,
[3H]UDP-Glc, and
UDP-[3H]Glc into Golgi vesicles was measured
exactly as described by Muñoz et al. (1996). One hundred
micrograms of protein was incubated with 1 µM
radiolabeled UDP-Glc (1 × 106 cpm/mL) at
25°C. The reaction was stopped by diluting with 10 volumes of
ice-cold STM buffer and filtering through 0.7-µm glass fiber filters
in a filtration system (model FH225V, Hoeffer, Piscataway, NJ).
Afterward, the filters were washed with an additional 10 volumes of
ice-cold STM buffer and dried, and the radioactivity was determined by
liquid-scintillation counting. The incorporation of
[3H]Glc into 70% ethanol or 10% TCA-insoluble
material was measured by incubating Golgi vesicles with
UDP-[3H]Glc exactly as described above, but the
reaction was stopped by adding ethanol or TCA to reach the appropriate
concentration. The samples were kept on ice and then filtered through
0.7-µm glass fiber filters. The filters were washed and dried, and
the radioactivity was determined by liquid-scintillation counting.
Analysis of Radiolabeled Metabolites Associated with the Golgi Vesicles or the Extravesicular Medium
Golgi vesicles (100 µg of protein) were incubated with [-32P]UDP-Glc for 10 min at 25°C. The
reaction was stopped by adding 10 volumes of STM buffer, and the
vesicles were immediately separated from the incubation medium by
filtration on 0.7-µm glass fiber filters using the filtration system
described above. The material not retained by the filters was collected
in tubes and an aliquot of this material was then analyzed by TLC and
autoradiography as described above. The radiolabeled metabolites
associated with the vesicles were extracted from them following the
procedure described by Waldman and Rudnick (1990). The vesicles
retained in the filters were boiled for 90 s in 1 mL of a solution
containing 1% SDS, 100 mM NaCl, 0.5 µM
UDP-Glc, 0.5 µM Pi, and 0.02%
NaN3. The samples were put on ice and aliquots
were immediately taken for analysis on TLC using PEI plates.
Enzyme and Protein Assays
UDPase in the presence and in the absence of 0.1% (v/v) Triton X-100 was measured as described by Briskin et al. (1987). Protein concentrations were measured by the BCA method (Pierce).
| |
RESULTS |
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|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
UDP-Glc is transported into the lumen of Golgi cisternae and
utilized for the synthesis of polysaccharides (Muñoz et al., 1996). This poses the question of whether UDP-Glc is accumulated and
concentrated in the lumen of the Golgi apparatus, as is the case in the
mammalian Golgi apparatus (Hirschberg and Snider, 1987), or if it is
immediately metabolized by glucosyltransferase(s). To address this
question we studied the transfer of [3H]Glc
onto endogenous acceptors after incubation of Golgi vesicles with
UDP-[3H]Glc. At different incubation times most
of the radioactivity incorporated from the transport of
UDP-[3H]Glc into Golgi vesicles could be
accounted for by the transfer of [3H]Glc into
ethanol- and TCA-insoluble material (Fig.
1). This result indicates that once in
the lumen of the Golgi apparatus, UDP-Glc is quickly metabolized and
the concentration of soluble UDP-Glc in the Golgi lumen is
undetectable.
|
UDP-Glc is transported to the lumen of Golgi cisternae and Glc is
transferred to endogenous acceptors, mainly polysaccharides, in a
reaction(s) catalyzed by glucosyltransferase(s). Based on the data
contained in this paper and information from previous work regarding
the transport of UDP-Glc (Muñoz et al., 1996
Received January 5, 1998;
accepted April 1, 1998.
Abbreviation:
PEI, polyethylene imide.
We would like to thank Lorena Norambuena for technical support,
Dr. Caroline Clairmont for reading the manuscript, Dr. Mario Mera
(Instituto de Investigaciones Agropecuarias-Carillanca, Temuco) for
providing the seeds, and the people from the Laboratory of Membranes,
Department of Biology, Faculty of Sciences, University of Chile, for
helpful discussions.
Abeijon C,
Robbins PW,
Hirschberg CB
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Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis.
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Feingold DS, Avigad G (1980) Sugar nucleotide transformations in
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Fredrikson K,
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Hirschberg CB,
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[Abstract]
Miura N,
Ishida N,
Hoshino M,
Yamauchi M,
Hara T,
Ayusawa D,
Kawakita M
(1996)
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Muñoz P,
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Orellana A
(1996)
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Orellana A,
Neckelmann G,
Norambuena L
(1997)
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or phosphate groups, and with 3H in the
uridine ring, and with these substrates we could follow the formation
of different metabolites generated during the metabolism of the
nucleotide in the Golgi vesicles. The possible radiolabeled products
that could be formed by the metabolism of these substrates are shown in
Figure 2. When Golgi vesicles were incubated with [-32P]UDP-Glc, the radiolabeled nucleotide
sugar decreased and [-32P]UMP increased,
accumulating with time (Fig. 3). Neither
[-32P]UDP nor 32Pi was
observed in this experiment. The absence of 32Pi
in this experiment suggests that [-32P]UMP
was not further hydrolyzed to uridine plus Pi. When
[-32P]UDP-Glc was used, we observed a
decrease in [-32P]UDP-Glc and an increase
and an accumulation of 32Pi with time (Fig. 3),
corroborating our previous results (Orellana et al., 1997). Again, no
[-32P]UDP was observed under this condition.
Moreover, in this work we could not detect
Glc-1-32P, a possible breakdown product of
UDP-Glc by organic pyrophosphatases, suggesting that the formation of
Pi and UMP did not occur by a mechanism mediated by Glc-1-phosphate.
This idea is also supported by the fact that we could not detect
phosphatase activity in the Golgi vesicles when Glc-1-P was used as a
substrate (Orellana et al., 1997).
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Figure 2.
UDP-Glc radiolabeled at various positions and the
potential resulting metabolic products for each. UDP-Glc radiolabeled
in the uridine ring with tritium (a), or with 32P in the
(b) and (c) phosphate groups, were prepared as described in
``Materials and Methods''. The possible radiolabeled products derived
from their metabolism are shown in each case.
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Figure 3.
Metabolism of [ -32P]UDP-Glc and
[-32P]UDP-Glc in pea stem Golgi vesicles
(100 µg of protein) incubated with 1 µM (1 µCi/nmol) of [-32P]UDP-Glc or [-32P]UDP-Glc.
After the indicated times an aliquot equivalent to 2 µg of protein
was taken, boiled for 1 min, and loaded on a PEI-TLC plate. The TLC
plate was run as described in "Materials and Methods," air-dried,
and exposed to 70°C with an enhancing screen. The migration of
standards is shown with arrows. The uridine derivative standard was
visualized by UV absorption and the Pi migration standard was
visualized using 32Pi.
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Figure 4.
Metabolism of [3H]UDP-Glc by pea
stem Golgi vesicles. Golgi vesicles (100 µg of protein)
were incubated with [3H]UDP-Glc (3 µCi/nmol) for 10 min
( 
). As a control, Golgi vesicles previously boiled for 3 min were
incubated under the same conditions (
). After 10 min an aliquot of
each sample equivalent to 2 µg of protein was taken, boiled for 1 min, and loaded onto a PEI-TLC plate. The plate was run, air-dried, and
cut into 0.5-cm fragments. The radioactivity associated with each
fragment was determined by liquid-scintillation counting. The migration
of standards is depicted.
). To test whether UMP was produced in the lumen
of Golgi vesicles, we proteolytically blocked the transport of UDP-Glc
into Golgi vesicles by a mild treatment with proteinase K under
conditions that do not disrupt the vesicles or affect the activity of
the enzymes located in the lumen (Muñoz et al., 1996; Orellana et
al., 1997). When the formation of UMP in the proteolyzed vesicles was
analyzed and compared with the control, we found a large decrease in
the formation of this metabolite (Fig. 5A). To ensure
that the decrease of the formation of UMP was due to a lack of UDP-Glc
transport into the Golgi vesicles and not to the proteolytic
inactivation of the enzymes involved in its formation, we permeabilized
the protease-treated Golgi vesicles to bypass the transport step. When
we did so, formation of UMP was restored, indicating that UMP was
formed in the lumen of Golgi vesicles and that UDP-Glc transport was
required. To demonstrate that proteolysis did not affect the activity
of lumenal enzymes we analyzed the topology of Golgi UDP- ase, a
lumenal enzyme, corroborating our previous results (Orellana et al.,
1997) (Fig. 5B).
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Figure 5.
Topology of UMP formation in Golgi vesicles. A,
Golgi vesicles (100 µg of protein) were incubated in the presence (+)
or absence ( ) of proteinase K (20 µg) for 30 min at 30°C. The
reaction was stopped with 1 mM PMSF, and then the vesicles
were incubated on ice for 10 min in the presence or in the absence of
0.1% Triton X-100. The vesicles were then incubated with 1 µM (1.0 µCi/nmol) [-32P]UDP-Glc for 10 min. The products formed during the incubation were analyzed by TLC
using PEI plates and exposed to autoradiography at 70°C with an
enhancing screen. Lanes: 1, Heat-inactivated Golgi vesicles; 2, normal
Golgi vesicles; 3, proteolyzed Golgi vesicles; and 4, Golgi vesicles
subjected to proteolysis and further permeabilization. Migration of
standards is shown by arrows. B, Topology of Golgi UDPase. Sealed or
Triton X-100-permeabilized Golgi vesicles were incubated in the
presence of proteinase K using the same conditions as described in A. The samples were then separated in native gels and the UDPase activity
was determined in situ as described in ``Materials and Methods''.
Lanes: 1, Untreated Golgi vesicles; 2, sealed Golgi vesicles treated
with proteinase K; and 3, permeabilized Golgi vesicles treated with
proteinase K. The arrowhead indicates the migration of Golgi UDPase in
the native gel.
-32P]UDP-Glc was rapid at the beginning but
that it reached equilibrium by 5 min. This behavior was similar to the
incorporation of [3H]UDP-Glc shown previously
(Muñoz et al., 1996). However, when [-32P]UDP-Glc was used, its incorporation
also reached a rapid equilibrium, although there was a slight
difference in the kinetic behavior compared with the other
UDP-Glc-radiolabeled substrates. These results suggest that the
metabolites of the nucleotide moiety of UDP-Glc generated in the lumen
of the vesicles were not accumulated in that compartment and that they
were transported out to the extravesicular medium. To further
investigate this point we analyzed at different times the content of
both the Golgi vesicles and the extravesicular medium after
incubation with [-32P]UDP-Glc. When the
vesicle content was analyzed, we detected only Pi, whereas UDP-Glc was
not detected at all (Fig. 7A). Radiolabeled 32Pi increased during the first 10 min and then
remained constant. No accumulation of Pi was detected in the vesicles.
When the extravesicular medium was analyzed, both UDP-Glc and Pi were
detected. UDP-Glc decreased with time, whereas Pi increased (Fig. 7B).
From this result and the result shown in Figure 5, we conclude that
Golgi vesicles incubated with [-32P]UDP-Glc
produced Pi in the lumen of the vesicles, which was then exported out
of the vesicular lumen.
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Figure 6.
Incorporation of [ -32P]UDP-Glc
and [-32P]UDP-Glc into pea stem Golgi
vesicles. Golgi vesicles (200 µg of protein) were
incubated with 1 µM (3.0 µCi/nmol) of
[-32P]UDP-Glc (A) or [-32P]UDP-Glc
(B) for different periods of time. The reaction was stopped by
filtration through 0.7-µm glass fiber filters, and the amount of
radioactivity associated with the filters was determined using
liquid-scintillation counting. The measurements were done in
triplicate, and the average and its deviation are depicted in the
figure. C, Incorporation of [3H]UDP-Glc into pea stem
Golgi vesicles from Muñoz et al. (1996).
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Figure 7.
Metabolites associated with Golgi vesicles upon
incubation with [ -32P]UDP-Glc. Golgi
vesicles (100 µg of protein) were incubated with 1 µM
(3.0 µCi/nmol) [-32P]UDP-Glc for different periods
of time. The incubation was stopped by filtration. A, The solutes
associated with the vesicles were obtained by solubilizing the material
associated with the filters and then analyzed by TLC on PEI plates. B,
The solutes not associated with the vesicles were collected from the
flow-through fraction of the filtration step and analyzed by TLC using
PEI plates. In both cases the plates were exposed to autoradiography at
70°C with an enhancing screen; however, the exposure times were
8 d in A and 2 d in B. The migration of standards is shown by
arrows.
-32P]UDP-Glc, we observed a decrease in the
radioactivity associated with the vesicles, suggesting that UDP-Glc
stimulated the efflux of UMP. In contrast, when
[-32P]UDP-Glc was used as a substrate and
32Pi was in the lumen, we did not see a decrease
in the radioactivity associated with the vesicles. A minor increase was
observed but different experiments indicated that it was not
significant. These results demonstrate that the radioactive metabolites
derived from the nucleotide moiety of UDP-Glc exit the vesicles by
different mechanisms. When the vesicles contained radiolabeled UMP, the efflux of this metabolite was stimulated by UDP-Glc, suggesting an
exchange between UDP-Glc and UMP by an antiporter mechanism. In
contrast, when the vesicles contained Pi, its efflux was not stimulated
by UDP-Glc, suggesting that Pi exits the vesicles by a mechanism
different from that of UMP.
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Figure 8.
UDP-Glc-induced efflux of radiolabeled solutes
associated with Golgi vesicles. Golgi vesicles (300 µg of protein)
were incubated for 10 min with 1 µM (3 µCi/nmol)
[ -32P]UDP-Glc (A), [-32P]UDP-Glc (B),
or [3H]UDP-Glc (C). After the incubation a pulse of cold
UDP-Glc (1 mM) was added to each sample (indicated with an
arrow), and the incubation continued for 0, 3, or 5 min. Finally, the
incubations were stopped by filtration, and the radioactivity that
remained associated with the vesicles after the pulse of UDP-Glc was
determined by liquid-scintillation counting. The amount of solutes
associated with the vesicles is expressed in equivalents of UDP-Glc at
the beginning of the incubation. The measurements were done in
triplicate and the average and its deviation are depicted in the
figure. The incorporation of the various substrates, obtained from
Figure 6, is depicted with a broken line.
DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References
) and the function of
Golgi UDPase (Orellana et al., 1997), our current working hypothesis on
UDP-Glc metabolism and polysaccharide biosynthesis in the Golgi
apparatus is depicted in Figure 9. According to this
model, the mechanism of the biosynthesis of Glc-containing polysaccharides in elongating tissues can be envisioned as the following cycle: biosynthesis and further transport of the nucleotide sugar into the Golgi cisternae, resulting in the incorporation of Glc
into macromolecules in the lumen of Golgi cisternae; the nucleotide
moiety is then quickly converted into UMP and Pi, metabolites that
return to the cytoplasm. One of the important concepts determined from
this model is that glycosyltransferases are not the only elements
involved in Glc-containing polysaccharide biosynthesis. The nucleotide
sugar transporters themselves could play an essential role in
polysaccharide biosynthesis by allowing the substrates to reach the
compartment where the transferases for polysaccharide biosynthesis are
located. Support for such a role has been provided in mammals, where
Madin-Darby canine kidney cells, mutants in a Golgi UDP-Gal
transporter, are unable to synthesize keratan sulfate, a galactosylated
proteoglycan (Toma et al., 1996). Whether the nucleotide sugar
transporter or the transferase is the limiting step for the rate of
polymer formation in plants is a question that still remains to be
answered.
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Figure 9.
Model for UDP-Glc metabolism in Golgi vesicles.
UDP-Glc is transported into the lumen of Golgi cisternae, Glc is
immediately transferred to polymers at different stages of growth, and
UDP is released. However, this product is immediately hydrolyzed by Golgi UDPase, producing UMP and Pi. Although a pool of both metabolites can be observed in the Golgi cisternae, they do not accumulate and
instead exit the Golgi cisternae. The exit of UMP is stimulated by
UDP-Glc, suggesting an exchange mechanism, whereas Pi exits the Golgi
cisternae by an independent mechanism.
). This
is in agreement with the fact that UMP and Pi are indeed produced in
the lumen of the Golgi vesicles. Moreover, UDPase is highly active in
the elongating region of pea stems, correlating with a high rate of
polysaccharide biosynthesis in the Golgi (Orellana et al., 1997).
). An
alternative explanation for the formation of UMP and Pi would be that
Glc transfer is mediated by a Glc-phosphate intermediate, followed by
its hydrolysis to release Pi. Although in previous work we have
detected in some cases low levels of Glc-1-P, these results are not
reproducible, and it changes from preparation to preparation. This is
in contrast to UMP and Pi formation, which are absolutely reproducible.
This suggests that the presence of Glc-1-P observed in some experiments
could be due to contamination of organic pyrophosphatase, which breaks down UDP-Glc (Feingold and Avigad, 1980). More importantly, we could
not detect any phosphatase activity toward Glc-1-P (Orellana et al.,
1997), suggesting that production of Pi from UDP-Glc does not involve a
Glc-phosphate intermediate. Taking all of this evidence together, we
favored a process mediated by UDPase to explain the formation of
UMP and Pi.
), we hypothesize
that the UDP-Glc transporter would correspond to a UDP-Glc/UMP
antiporter. Since UDP-Glc is rapidly metabolized, a constant influx of
this substrate into the lumen is required. Therefore, it would be
necessary to have a rapid and constant supply of UMP in the lumen. This
would be provided by the hydrolysis of UDP catalyzed by UDPase. From
this point of view, this enzyme plays a critical role in the mechanism because it drives the transfer reaction by consuming UDP, eliminates an
inhibitor for glucosyltransferases, and provides UMP as the substrate
for the putative antiporter, allowing the entrance of additional
UDP-Glc. Thus, UDPase would play an important role in polysaccharide
biosynthesis, explaining at the same time the high activity of this
enzyme in the elongating region of a growing plant (Orellana et al.,
1997).
). In this work we have
demonstrated that the [3H]uridine-containing
nucleotide corresponds to UMP; therefore, the efflux of UMP is
specifically stimulated by uridine-derivative nucleotide sugars
involved in polysaccharide biosynthesis in the Golgi. Since many of the
substrates required for polysaccharide biosynthesis are uridine-derived
nucleotide sugars, and since an active Golgi UDPase is found in the
lumen, the supply of UMP and Pi to their respective lumenal pools may
occur from a number of substrates used in both hemicellulose and pectin
biosynthesis. Therefore, it is possible to postulate that many, if not
all, nucleotide sugars that are transported into the lumen of Golgi cisternae may use an antiporter mechanism.
; Eckhardt
et al., 1996; Miura et al., 1996; Ma et al., 1997), as well as a Golgi
GDPase in yeast (Abeijon et al., 1993); unfortunately, no homologous
genes in plants have been described so far. The cloning of nucleotide
sugar transporters and Golgi UDPase in plants will be an important tool
to test this model.
1
This work was supported by grant no. 1970494 from Fondecyt (Chile).
FOOTNOTES
*
Corresponding author; e-mail aorellan{at}abello.dic.uchile.cl; fax
56-2-271-2983.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-D-glucan synthase activity from pea tissue.
Eur J Biochem
220:
943-953
[Web of Science][Medline] -glucan synthase.
Biochem Soc Trans
20:
710-713
[Web of Science][Medline] -diphosphatase from pea stems.
Plant Physiol
114:
99-107
[Abstract] -glucan synthase particles from plant cells and identification with Golgi membranes.
Proc Natl Acad Sci USA
64:
605-612
Copyright Clearance Center: 0032-0889/98/117/1007/08
© 1998 American Society of Plant Physiologists
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-Diphosphate-Glucose in
Golgi
Vesicles from Pea Stems
), or by adding ethanol (
).
The results are expressed as the percentage of the total radioactivity
in UDP-[3H]Glc initially present in the assay. The
experiment was repeated three times in duplicate, and the average
values and their deviations are plotted. A representative experiment is
shown.