Journal of Experimental Botany, Vol. 48, No. 311, pp. 1209-1214, June 1997
Journal of
Experimental
Botany
Distribution of xylosyltransferases and
glucuronyltransferase within the Golgi apparatus in
etiolated pea [Pisum sativum L.) epicotyls
E.A-H. Baydoun1 and C.T. Brett 23
1
Department of Biology, American University of Beirut, Beirut, Lebanon
2
Plant Molecular Science Group, Institute of Biomedical and Life Sciences, University of Glasgow, UK
Received 30 September 1996; Accepted 19 February, 1997
Abstract
Membranes from etiolated pea epicotyls were fractionated by discontinuous sucrose-density-gradient
centrifugation into endoplasmic reticulum, Golgi
apparatus and plasma membrane. The Golgi apparatus
was further fractionated on a shallow, continuous sucrose density gradient into Golgi subtractions of low,
medium and high density. Xylosyl- and glucuronyltransferases were both found to be concentrated in
the Golgi apparatus. The glucuronyltransferase was
concentrated in the low-density Golgi membranes, but
xylosyltransferases were found in all three Golgi
subfractions. The multiple location of xylosyltransferases within the Golgi was found in both younger and
older regions of the epicotyl, and xylan was the major
product of the xylosyltransferase in the low- and
medium-density subfractions. In the presence of
UDPglucose, xylose was also incorporated into xyloglucan, but this activity was concentrated in the highdensity Golgi membranes.
Key words: Hemicellulose biosynthesis, xylan, xyloglucan,
Golgi apparatus.
Introduction
Cell wall matrix polysaccharides are synthesized in the
Golgi apparatus and then secreted into the wall (Brett
and Waldron, 1996). Two of these matrix polysaccharides,
the xylans and the xyloglucans, contain xylose as a major
component. The xylans are a group of matrix polysaccha-
rides which contain an acetylated /3-1,4-linked xylan backbone with single 4-<9-methylglucuronic acid and arabinose
residues as side-chains. The frequency of these side-chains
varies in different species and cell types, and xylans are
often referred to as arabinoxylans, glucuronoarabinoxylans or glucuronoxylans, depending on the relative
amounts of the two types of side-chain. The xyloglucans
contain a backbone of /9-1,4-linked glucose residues, to
the majority of which xylose residues are attached as sidechains by a(l-6)-linkages; some of the xyloses are substituted with galactose or fucosylgalactose.
Both xylans and xyloglucans are synthesized by the
co-operative action of several glycosyltransferases, and in
the case of the xylose residues the sugar donor is uridinediphosphate-xylose (UDPXyl). The subcellular localization of the UDPXyl: xyloglucan xylosyltransferase has
been studied, and the enzyme is predominantly located
in the Golgi apparatus (Brummell et al., 1990).
Immunocytochemical studies also indicate that xyloglucan formation occurs in this organelle (Staehelin and
Moore, 1995). Indirect evidence from cell fractionation
studies suggests that the UDPXyl: xylan xylosyltransferase is also located in the Golgi apparatus (Waldron and
Brett, 1987).
The Golgi apparatus from etiolated pea seedling stems
was separated into three subfractions of low-, mediumand high-density by density-gradient centrifugation, and
it was shown that the glucuronyltransferase (GT)
involved in glucuronoxylan biosynthesis is associated
chiefly with the low-density Golgi membranes (Hobbs
et al., 1991). The same Golgi subfractionation procedure
is now used to investigate the distribution of xylosyltransferases (XT) within the Golgi apparatus.
'To whom correspondence should be addressed at: Bower Building, University of Glasgow, Glasgow G12 8QQ, UK. Fax: +44 141 330 4447. E-mail:
cbrettebio.glaac.uk
6 Oxford University Press 1997
1210 Baydoun and Brett
Materials and methods
Driselase digestion
Peas (Pisum sativum L. cv. Alaska and Meteor) were obtained
from Sharpes International, Sleaford, UK. The Alaska cultivar
was used for the majority of the experiments; this became
unavailable towards the end of the work. No differences were
detected between the two varieties with respect to the membrane
fractionation or enzyme localization. Seedlings were grown in
the dark for 5 d on moist vermiculite at 25 °C.
Insoluble products of XT assays were incubated with 100 fi\
driselase (2.5%) (Sigma) in pyridine (1%) - acetic acid (1%) for
40 h at 25 °C (Fry, 1988). Digestion products were run on
paper chromatography on Whatman No. 1 paper in butanol/
acetic acid/water (12:3:5, by vol.) for 30 h. The paper was cut
into 1 cm strips for scintillation counting. In some incubations,
the driselase was supplemented with jS-xylosidase (0.02 units,
Sigma), and the digestion and analysis carried out in the
same way.
Membrane fractionation
Chemicals
Membranes were fractionated as described (Hobbs et al., 1991).
Briefly, epicotyl tissue (lOOg) was chopped with a razor blade
and then homogenized in a pestle and mortar with cold 10 mM
TRIS-HC1 (pH7.4) containing 10 mM KC1, 1.5 mM MgCl2
and 10 mM dithiothreitol. The homogenate was strained
through four layers of muslin. The residue was rehomogenized
in the homogenization buffer and strained, and compensation
buffer (10 mM TRIS-HC1 (pH 7.4) containing 0.72 M KC1,
28.5 mM MgCl2, 10 mM dithiothreitol, and 2 M sucrose) added
to give a sucrose density of 1.032 gem" 3 . The filtrate was
centrifuged at 13 000 g for lOmin. The supernatant was
centrifuged on to a cushion of 40% (w/w) sucrose (composition
shown below) at 100 000 g for 1 h. For certain experiments, the
membranes which collected at the interface were pelleted by
centrifugation at 100 000 g for 1 h to form the 'general
membrane fraction'. For fractionation into endoplasmic reticulum, Golgi apparatus and plasma membrane, the membranes
at the interface were collected and adjusted with a sucrose
solution (lOmM TRIS-HC1 (pH 7.4), 5 mM MgCl2, 100 mM
KC1, 10 mM dithiothreitol, and 2.26 M sucrose) to a final
sucrose concentration of 40% (w/w). A discontinuous sucrose
gradient was formed on top of the membranes and consisted of
35, 25 and 18% (w/w) sucrose in 40 mM TRIS-HC1 (pH 7.4)
containing 0.1 mM MgCl2, 1 mM EDTA and 10 mM dithiothreitol. The four 'steps' have densities of 1.179, 1.165, 1.115,
and 1.074 g cm"3, respectively. After centrifugation at 100000 g
for 4 h, fractions obtained at the interfaces were enriched in
endoplasmic reticulum (18/25%), Golgi apparatus (25/35%) and
plasma membrane (35/40%) (Hobbs et al., 1991). The Golgi
apparatus was further fractionated by centrifugation on a
continuous sucrose density gradient (1.105-1.155 g cm"3) at
100 000 g for 16 h, to separate the three Golgi subfractions
(Hobbs etal., 1991).
Radioactive chemicals were obtained from NEN Dupont,
Wedgwood Way, Stevenage, Herts SGI 4QN, UK. Nonradioactive sugar-nucleotides were obtained from Sigma-Aldrich
(UK), Fancy Road, Poole BH12 4QH, UK.
Plant material
Enzyme assays
Incubations (25 °C, 1 h) for the assay of glucuronyltransferase
(GT) contained UDP-(U-14C)GlcA (460 Bq, 0.5 pM), UDPXyl
(1.0 mM), MnCl2 (lOmM), and membranes (50^1) in a total
volume of 100 pi. Incubations (25 °C, 1 h) for the assay of
xylosyltransferase (XT) contained UDP-{U-14C)Xyl (8.3 kBq,
made up to 0.1 mM by the addition of non-radioactive
UDPXyl), MnCl2 (10 mM) and membranes (50 /il) in a total
volume of 100^1. In each case incubations were terminated
with 70% ethanol (1 ml), and the insoluble material washed
three times with 70% ethanol (1 ml) and once with water
(1 ml). Radioactivity in the washed pellet was determined by
liquid scintillation counting. Incorporation of radioactivity into
polysaccharide by both XT and GT has been shown to be
linear over the 1 h incubation period under the conditions used
(Baydoun et al., 1989; Waldron and Brett, 1983). Latent inosine
diphosphatase (IDPase), used as a marker for Golgi membranes,
was assayed as described by Hobbs et al. (1991).
Results
Distribution of XT and GT between the endoplasmic
reticulum (ER), Golgi apparatus (GA) and plasma membrane
(PM)
ER, GA and PM fractions were separated on a discontinuous sucrose density gradient and assayed for XT and GT
activity. Both enzymes were found to be concentrated in
the GA (Table 1). However, the observed variation in
activity between the three membrane fractions could have
been partly due to variations in the levels of permeases
for the sugar-nucleotide substrates, since glycosyltransferases in the endomembrane system are thought to be on
the luminal side of the membrane. The activity of each
enzyme was therefore tested in the presence and absence
of Triton X-100 (0.1%, w/v), which would be expected to
make the membranes permeable to the substrates
(Table 2). The experiment was carried out at two different
MnCl2 concentrations, 10 and 1 mM, which are the
optima for paniculate and Triton-X-100-solubilized GT,
respectively (Waldron et al., 1989). At both concentrations, Triton X-100 decreased the incorporation of both
radioactive sugars into polysaccharide, indicating that
access of substrates to enzymes was probably not a
limiting factor. Hence it can be concluded that both GT
and XT are localized chiefly in the Golgi apparatus.
Table 1. Activity of xylosyltransferase and glucuronyltransferase
in the endoplasmic reticulum, Golgi apparatus and plasma
membrane
Enzyme activity
(Bq h " 1 g " 1 fr. wt. x 101)
Endoplasmic reticulum
Golgi apparatus
Plasma membrane
XT
GT
3.4
30.4
5.4
32.6
3.0
7.6
Golgi xylosyl- and glucuronyltransferases
Table 2. Effect of Triton X-100 on xylosyltransferase (XT) and
glucuronyltransferase (GT) in the general membrane fraction
Enzyme
MnCl2
(mM)
Tnton X-100
(0.1% w/v)
Enzyme activity
( B q h - ' g " 1 fr. wt. x 102)
XT
XT
XT
XT
GT
GT
GT
GT
10
10
1
1
10
10
1
1
+
—
+
—
+
—
+
28.8
13.6
32.0
11.2
42.4
36.8
69.6
42.4
Distribution ofGT and XT within the Golgi apparatus
The Golgi-enriched fraction from the discontinuous sucrose density gradient was further fractionated on a shallow continuous density gradient to separate the Golgi
subfractions. The low-, medium- and high-density Golgi
subfractions are found at densities of 1.122, 1.135 and
1.146 g cm"3, respectively (Hobbs et al., 1991). GT was
found mainly in the low-density region of the gradient,
while XT was found in all three regions (Fig. 1). The
apparent multiple location of XT might have been due
to epimerization of UDPXyl to UDPAra, followed by
incorporation of radioactive arabinose into polysaccharide, in some of the fractions. To check this, XT incubations were carried out using pooled fractions from the
three regions of the gradient. The polysaccharide products
were hydrolysed with trifluoroacteic acid and the radioactive sugars separated as described by Baydoun et al.
(1989). In each case more than 85% of the radioactivity
was found in xylose, and less than 15% in arabinose,
confirming that XT is present in all three Golgi
subfractions.
Low
1211
Differences between different regions of the epicotyl
One possible explanation for the multiple location of XT
within the Golgi apparatus was that primary and secondary wall xylans, which differ in some details of their
structure (Brett and Waldron, 1996), might be made in
different parts of the Golgi. Assay of the tranferases in
six epicotyl regions of increasing maturity showed that
XT and GT were both present in all regions of the
epicotyl (Table 3). Golgi membranes from the youngest
and oldest epicotyl sections were compared by continuous
density gradient centrifugation (Fig. 2a, b). Both sections
contained XT activity in regions of the gradient corresponding to all three subfractions; the main difference
between them was a relatively high XT activity in the
medium-density subfraction in the older tissue compared
to the younger tissue. There were also differences in the
GT distribution, the younger tissues showing a greater
amount of GT in the medium-density Golgi subfraction.
It was clear from these results that the multiple location
of XT in all three subfractions was unlikely to be due to
Table 3. Specific activities of xylosyl- and glucuronyltranferase
in 1 cm sections of the epicotyl
Distance from hook (cm)
Medium
050
Enzyme activity
(Bq h " ' mg"' protein)
XT
GT
4.5
4.3
3.9
5.4
4.9
5.3
19.8
21.7
7.6
8.8
10.8
6.3
High
r-1.18
wei
inco rpo
^3 ft,
I
1 025
_*-
1.14 3
'an
Kadi
3
Q
i
1.10
Fraction
Fig. 1. Golgi membranes from the whole epicotyl were fractionated by continuous sucrose density gradient centrifugation, and fractions assayed
for XT (closed squares) and GT (open squares). Densities of the fractions were also measured (closed circles). The approximate positions of the
low-, medium- and high-density Golgi membranes are indicated, by comparison of the fraction densities with Fig. 2 of Hobbs et al. (1991).
1212 Baydoun and Brett
5.0
2
1.18
r0.2
M
if
s
"8
incorpo
e
it)
50
M
%
Jr 2-5
u
9
'ati
__ •
5
I
0J
Fraction
Fig. 2. Golgi membranes from the youngest (a) and oldest (b) 1 cm-sections of the epicotyl were fractionated by continuous sucrose density
gradient centrifugation, and fractions assayed for XT (closed squares), GT (open squares) and latent IDPase (open triangles). Densities of the
fractions were also measured (closed circles).
a differential localization of the enzymes for primary and
secondary wall xylan synthesis.
Differential localization of xylan and xyloglucan synthesis
within the Golgi apparatus
Another possible explanation for the multiple location of
XT within the Golgi is that the enzymes involved in the
synthesis of both xylan and xyloglucan are being
observed, even though relatively little xyloglucan would
be expected to be formed in the absence of UDPGlc
(which is needed to form the glucan backbone; Campbell
et ai, 1988). Incubation of the polysaccharide products
from each Golgi region with xylanase, as described by
Baydoun et al. (1989), solubilized radioactive material
from all three subfractions in both young and old regions
of the epicotyl (results not shown), indicating that radioactive xylan was formed in all three subfractions.
When UDPGlc (0.1 mM) was included in the XT
assay, incorporation of radioactive xylose into poly-
saccharide was inhibited in the low- and medium-density
Golgi membranes, but stimulated in the high-density
subfraction (Table 4). Digestion of the radioactive products with driselase (Fry, 1988) produced radioactive digestion products which ran on paper chromatography with
an R^ of 0.4 to 0.5, corresponding to the mobility of the
disaccharides xylobiose (Xyl-/?(l-4)-Xyl) and isoprimeverose (Xyl-a(l-6)-Glc) (Fry, 1988), together with products running with xylose markers. Xylobiose and
Table 4. Activity of xylosyltransferase in the presence and absence
of UDPGlc (0 1 mM) in the Golgi subfractions
Golgi subfraction
Low density
Medium density
High density
Xylosyltransferase activity
(Bq h" 1 g" 1 fr. wt. x 102)
-UDPGlc
+ UDPGlc
44.2
29.0
5.8
27.0
21.4
8.2
Golgi xylosyl- and glucuronyltransferases
isoprimeverose are expected to be produced by enzymic
hydrolysis of xylan and xyloglucan, respectively. To distinguish between these two polysaccharides, the driselase
was supplemented with /9-xyIosidase, and the digestion
repeated. Under these conditions, disaccharide digestion
products were only observed in the case of the highdensity Golgi membranes, and only when these membranes had been incubated in the presence of UDPGlc.
In this case, 70% of the products of the XT incubation
were converted to disaccharide, and 30% to xylose. Betaxylosidase would be expected to hydrolyse xylobiose, but
not the alpha-linked isoprimeverose, and since driselase
is known to lacka-xylosidase (Fry, 1988), the disaccharide
produced in the presence of driselase and /J-xylosidase is
likely to be isoprimeverose, derived from xyloglucan.
These results confirm that the XT present in the highdensity Golgi membranes is concerned mainly with the
production of xyloglucan rather than xylan, and that this
region of the Golgi apparatus is the major site of incorporation of xylose into xyloglucan.
Discussion
These studies provide the first direct evidence that the
xylosyltransferase responsible for the incorporation of
xylose from UDPXyl into xylan is localized chiefly within
the Golgi apparatus. They also indicate that the enzyme
is present in all three Golgi subfractions, with the lowand medium-density Golgi membranes containing more
than the high-density Golgi membranes. This distribution
is not greatly different from that of the glucuronyltransferase which adds the glucuronic acid side-chains, which is
concentrated chiefly in the low-density Golgi membranes,
with smaller amounts in other subfractions. However, the
XT:GT ratio does vary in the three subfractions, and the
peaks of XT and GT in the continuous gradients do not
correspond well (Figs 1,2). This suggests either that the
degree of glucuronidation of the xylan is controlled by
the XT:GT ratio in rather a complex way, or that the
XT:GT ratio is not the most important factor determining
the degree of glucuronidation. Structural studies of the
glucuronidation pattern in legumes and other plants suggest considerable uniformity in the pattern (Nishitani and
Nevins, 1991), which would point to the XT:GT ratio
not being the determining factor.
The stimulation of XT by UDPGlc to form xyloglucan
in the high-density Golgi membranes, but not in the other
subfractions, indicates that the xylosyltransferase responsible for incorporating xylose into xyloglucan is concentrated in the denser part of the Golgi. This is in accordance
with immunocytochemical observations, which suggest
that xyloglucan is synthesized chiefly in the trans-Golgj
cisternae and trans-Go\gi network (Staehelin and Moore,
1995). It is also in accordance with the membrane fractionation results of Brummell et al. (1990), who found
1213
the majority of the UDPXyl: xyloglucan xylosyltransferase in the more dense parts of the Golgi apparatus.
Taken together, these results indicate that xylan and
xyloglucan synthesis are largely separated into the earlier
and later stages, respectively, of the Golgi membrane flow
system. This finding conforms to the general pattern of
differential distribution of glycosyltransferases amongst
the subcompartments of the Golgi apparatus (Staehelin
and Moore, 1995).
Comparison of the results of Staehelin's group
(Staehelin and Moore, 1995) with the present work
indicates that the high-density Golgi membrane fraction
reported here may correspond to the trans-Golgi subcompartment observed by Staehelin's group, since both
appear to be the site of xylose incorporation into xyloglucan. In that case, the low- and medium-density membrane fractions may correspond to the cis and medial
Golgi subcompartments. However, distribution of at least
some biosynthetic enzymes within the Golgi apparatus
varies in different cell types (Lynch and Staehelin, 1992),
so confirmation that the three Golgi subfractions reported
here represent the cis, medial and trans Golgi may require
a co-ordinated cell-fractionation and immunocytochemical study on a single cell-type.
Acknowledgement
We thank the Research Board of the American University of
Beirut for financial support.
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Baydoun and Brett
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