Download Effect of Brefeldin A on the synthesis and transport of cell wall

Journal of Experimental Botany, Vol. 48, No. 316, pp. 1925-1933, November 1997
Journal of
Experimental
Botany
Effect of Brefeldin A on the synthesis and transport
of cell wall polysaccharides and proteins in
pea root seedlings
Remigio Lanubile, Gabriella Piro and Giuseppe Dalessandro1
Dipartimento di Biologia, Universita di Lecce, via prov.le Lecce-Monteroni, 1-73100 Lecce, Italy
Received 9 May 1997; Accepted 18 June 1997
Abstract
The in vivo effect of Brefeldin A (BFA) on the synthesis
and transport of cell wall polysaccharides and proteins
in the roots of pea seedlings (Pisum sativum L. cv.
Alaska) was investigated. BFA (10 //gml" 1 ) inhibited
the synthesis of cell wall matrix polysaccharides by
approximately 43%. Under the same conditions, cellulose synthesis was inhibited by approximately 77%.
The percentage of incorporation of L-[U-14C]leucine
and L-[U-14C]proline into cytosolic, membrane and cell
wall proteins was only slightly changed in the presence
of BFA. In addition, the drug did not change the pattern
of newly synthesized proteins in the three fractions as
judged by SDS-PAGE fluorography. Double labelling of
proteins and cell wall polysaccharides confirmed the
above reported data. All these results showed that the
synthesis and transport of proteins to the cell wall
was only slightly affected by BFA under similar conditions to those which brought about a strong inhibition
of the synthesis of matrix and cellulosic polysaccharides. BFA had no effect on the activity of membranebound and digitonin-solubilized mannan and glucomannan synthase isolated from the third internode of
pea seedlings. This would exclude an effect of BFA at
the level of the catalytic site of the synthases. The
inhibition of polysaccharide synthesis by the drug was
rapidly eliminated after its removal. It is concluded
that the effect of BFA on the biosynthesis of cell wall
polysaccharides could be caused by an interaction of
the drug with the topological organization of the synthase complexes in the membranes. This effect would
precede the action of the drug at the level of vesicle
transport to the walls.
1
Key words: Brefeldin A, cell wall polysaccharides (synthesis
and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport).
Introduction
The Golgi bodies of both animal and plant cells are
dynamic organelles which play a key role in processing,
maturation and sorting of newly synthesized proteins,
glycoproteins and proteoglycans. These are transported
to specific membrane domains or they are secreted from
the cell. The Golgi bodies are also involved in the recycling
of receptors during endocytosis. In plant cells the Golgi
apparatus is the site of synthesis and packaging of cell
wall matrix polysaccharides such as pectins and hemicelluloses (Northcote, 1985). Biochemical and immunocytochemical studies have shown that the synthesis of matrix
polysaccharides occurs in different cisternae of the Golgi
stacks before they are packaged into secretory vesicles.
Vesicles take part in the intra-Golgi transport of the
polymers in a cis-to-lrcins Golgi direction (Zhang and
Staehelin, 1992; Staehelin and Moore, 1995). Cellulose is
polymerized at the plasma membrane. The enzymic complexes required for the synthesis of pectins, hemicelluloses,
and cellulose are synthesized, at least in part, on the
endoplasmic reticulum and then transported and sorted
to the Golgi cisternae. Cellulose synthase complex is
transported to the plasma membrane by secretory vesicles.
Thus, the dynamic structural and functional organization
of the plant cell wall during growth and differentiation
depends on the activities of the enzymes associated with
the endomembrane system.
Brefeldin A (BFA), a macrocyclic hydrophobic lactone
isolated from a variety of fungi (Harri et ai, 1963; Betina,
To whom correspondence should be addressed. Fax: +39 832 320626. E-mail: [email protected].
Abbreviations : ARF1, ADP-ribosylation factor 1; BFA, Brefeldin A.
Oxford University Press 1997
1884
Miller et al.
v_
c -;.-». _.. .i'v
Fig. 2. Electron micrograph of a growing root hair of Equisetum hyemale showing Golgi vesicles in the hemisphere of the tip and Golgi bodies
(GB) subapically. Bar= 1 ^m.
towards the edge of the pollen tube when nearing the
vesicle-rich region and are absent from the vesicle-rich
region proper (Miller et al., 1996). Treatment with
caffeine, a substance that greatly diminishes the calcium
influx and abolishes the tip-focused calcium gradient
(Pierson et al., 1996), causes the actin filaments to redistribute in such a way that they are closer to the tip,
thereby shortening the vesicle-rich region. When caffeine
is removed, the vesicle-rich region re-establishes its normal
size simultaneously with the removal of actin filaments
from this area (Miller et al., 1996). For further discussion
of pollen tube actin, see Miller et al. (1996). Since pollen
tubes and root hairs are both polar growing cells with
similar morphology, organelle distribution and cytoplasmic streaming patterns, it would seem logical that the
actin distribution within growing root hairs is similar to
that of a pollen tube. Future experiments should address
this issue.
Spectrin-like antigen
Animal spectrins are multifunctional molecules that
belong to a family of proteins including actin binding
protein 1, a-actinin, dystrophin, and fimbrin. These proteins have many binding sites with at least two actin
filament binding sites, and binding sites for calcium and
calmodulin (Hartwig, 1994). Spectrin and its associated
proteins in red blood cells are now thought to provide
organizational stability to a cell by controlling integral
membrane protein distribution (Bennett and Gilligan,
1993; Devarajan and Morrow, 1996). Based on data from
adrenal chromaffin cell secretion Hays et al. (1994) suggest that an increase of cytosolic calcium concentration
([Ca2+]c) dissociates actin from spectrin thereby allowing
vesicle fusion.
Immunoblot analysis of young tomato leaves detected
a 220 kDa protein labelled with human erythrocytespectrin (Michaud et al., 1991), and Faraday and
Spanswick (1993) detected a 230 kDa protein with antispectrin in the purified plasma membrane fraction of rice
roots. In addition, De Ruijter and Emons (1993) detected
spectrin-like epitopes in several tissues of maize and
carrot, predominantly at the plasma membrane of cells
growing along their whole length. This spectrin-like epitope is also localized at the tip of growing root hairs
(Fig. 3), which is comparable to its localization at the
tips of tobacco pollen tubes (Derksen et al., 19956) and
the tips of growing hyphae of Saprolegnia ferax
(Kaminskyj and Heath, 1995). Even though the nature
of the plant epitope found in root hairs is still not known,
its location suggests a role in exocytosis.
Calcium gradient
External Ca2+ ions at the appropriate concentration and
a Ca2+ influx are required to elicit exocytosis in
Arabidopsis root hairs (Schiefelbein et al., 1992). These
authors observed a net influx of Ca2+ at the tips of
growing root hairs with a calcium selective vibrating
probe, whereas no influx was found at the sides of the
hair nor into non-growing hairs. Selective vibrating probe
analysis indicated Ca2+ currents only in the tips of
growing Sinapis root hairs (Herrmann and Felle, 1995;
Felle and Hepler, 1997), which responded to changes in
external concentrations of calcium by transient growth
differences rather than an altered steady-state growth rate
(Herrmann and Felle, 1995). The [Ca2+]c increased in the
apical area in the presence of increased external calcium
and decreased when the external calcium concentration
was lowered (Felle and Hepler, 1997). In the latter study
non-growing hairs responded to changes in external calcium as well, but the [Ca2+]c increase was uniform
1926
Lanubile et al.
1992), has been widely used in animal cells to investigate
the transport of proteins and glycoproteins along the
secretory pathway and also the structure and function of
the Golgi apparatus. It causes numerous effects such as
(a) the inhibition of vesicular transport of membranes
and secretory proteins between the endoplasmic reticulum
and Golgi and intra-Golgi transport (Misumi et al., 1986;
Oda et al., 1987); (b) the rapid disassembly of the Golgi
apparatus and the redistribution of Golgi proteins and
lipids into the endoplasmic reticulum by a mechanism
which requires energy and microtubules (Fujiwara et al.,
1988; Domse/a/., 1989; Lippincott-Schwartz e? al., 1990);
(c) the release of the non-clathrin coated protein jS-COP
from the Golgi apparatus (Donaldson et al., 1990);
(d) changes in the morphology and function of other
components of the endomembrane system such as endosomes, lysosomes and the trans Golgi network (TGN)
(Lippincott-Schwartz et al., 1991; Klausner et al., 1992);
(e) the stimulation of the synthesis of sphingomyelin,
glucosylceramide and cholesterol ester and at the same
time the inhibition of the synthesis of cholesterol, triacylglycerol and phosphatidyl-choline (Kallen et al., 1993);
(f) the alteration of synthesis and processing of O- and
TV-linked glycans (Chawla and Hughes, 1991; Collins and
Mottet, 1992). BFA appears to cause most of its effects
at the Golgi apparatus by inhibiting the activation of
ADP-ribosylation factor 1 (ARF1) catalysed by the
Golgi-associated GTP/GDP exchange protein. This prevents the binding of ARF1 and coatomer, a cytosolic
coat protein complex, to Golgi membranes (Orci et al.,
1991; Donaldson et al., 1992; Helms and Rothman, 1992).
Thus ARF1, or an ARF-activating protein (Randazzo
et al., 1993), seem to be the key BFA-sensitive component
required for COPs binding to Golgi membranes. All
morphological and physiological effects of BFA disappear
when the drug is removed from the cells.
Recently, BFA has been used to elucidate the morphological and functional changes of the plant Golgi apparatus as well as other components of the endomembrane
system. Ultrastructural (Satiat-Jeunemaitre and Hawes,
1992; Rutten and Knuiman, 1993; Yasuhara et al., 1995;
Gorbatenko and Hakman, 1995), immunocytochemical
(Satiat-Jeunemaitre and Hawes, 1993; Henderson et al.,
1994; Satiat-Jeunemaitre et al., 1996) and biochemical
(Holwerda et al., 1992; Driouich et al., 1993; Jones and
Herman, 1993; Gomez and Chrispeels, 1993; Schindler
et al., 1994; Kunze et al., 1995) investigations on different
plant materials have confirmed that many aspects of BFA
effects on plant cells are similar to those reported in
animal cells. In particular, secretion of soluble proteins
to the vacuole (Driouich et al., 1993; Holwerda et al.,
1992; Jones and Herman, 1993; Gomez and Chrispeels,
1993) and matrix polysaccharides such as pectins and
hemicelluloses to the wall (Driouich et al., 1993) are
inhibited by BFA whereas the drug does not interfere
with the transport of the tonoplast integral protein (aTIP) (Gomez and Chrispeels, 1993). These results have
indicated that proteins may follow different pathways to
the same destination. It has also been reported that low
concentrations of BFA modify secretion and glycosylation
of secretory proteins without any breakdown of the Golgi
stacks (Driouich et al., 1993; Gomez and Chrispeels,
1993; Kimura et al., 1993).
In the present study the in vivo effects of BFA on the
synthesis and transport of cell wall polysaccharides and
proteins in the roots of pea seedlings (Pisum sathntm L.
cv. Alaska) were investigated. The results obtained demonstrate that BFA rapidly inhibits the biosynthesis of
cellulose and matrix polysaccharides whereas the synthesis
and transport of proteins to the cell wall remain
unchanged.
Materials and methods
Chemicals and radiochemicals
Brefeldin A, GDP-D-mannose, GDP-D-glucose and all other
chemicals were purchased from Sigma Chemical Co., St Louis,
Mo., USA. D-[U-14C]glucose (specific activity 10.5 GBq
mmol" 1 ), L-[U-14C]proline (specific activity 9.32 GBq mmol" 1 ),
L-[U-14C]leucine (specific activity 11.7 GBq mmol"'), L-[4,53
H]leucine (specific activity 3.15 TBq mmol" 1 ), L-[35S]methionine (specific activity >37 TBq mmol" 1 ), GDP-[U- 14 C]glucose
(specific activity 8.40 GBq mmol" 1 ), GDP-[U- 14 C]mannose
(specific activity 11.2 GBq mmol" 1 ) were from Amersham
International, Amersham, Bucks, UK..
Plant material and radiolabellmg expenments
Seeds of Pisum sativum L. cv. Alaska were soaked in running
tap water (8 h), planted in damp vermiculite and grown for 2 d
at 25±1°C in darkness. Using a dim green light, uniform
seedlings having primary roots 20-22 mm long were carefully
washed in sterile bidistilled water and incubated with or without
different concentrations of BFA in the presence of the
radioactive tracers as specified for each experimental condition.
The uptake of the radioactive tracer represents the difference
between the initial radioactivity of the incubation mixture and
the radioactivity remained at the end of incubation period of
the seedlings.
Radioactive cell wall polysaccharide labelling
Three pea seedlings were incubated in water with or without
BFA in the presence of 4 ^Ci of r>[U- 14 C]glucose at 25 "C, in
the dark, as specified for each experiment in tables and figures.
At the end of the radiolabelling period, the roots were rapidly
washed with an ice-cold unlabelled tracer solution and the root
cap removed. Starting from the root apex, segments 1 cm long
were cut from each root and homogenized with 2 ml Tenbroeck
tissue grinder in 10 mM sodium-phosphate buffer pH 7.2,
containing 10 mM imidazole, 1 mM benzamidine, 5 mM
6-amino-esanoic acid, 10 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The final volume of the
homogenate was measured and aliquots were counted for
radioactivity in order to obtain the total amount of the
radioactive material present in each homogenate. The homogenates were centrifuged at 800 x g for 10 min at 2 =C in a model
J2 21 centrifuge, rotor head J-20 (Beckman Instruments, Inc.,
BFA: cell wall and protein synthesis
Palo Alto, California, USA). The pellets, consisting of cell
walls, were incubated in 0.05% sodium deoxycholate for 2 h, at
20 °C and then washed with the homogenization buffer (twice)
and acetone (twice) to obtain purified cell walls. The 800 xg
supernatant was centrifuged at lOOOOOxg for 60 min at 2°C in
a Beckman preparative ultracentrifuge (model L8-55, rotor
head SW 55) to obtain a supernatant (100000 x g SN) and a
pellet consisting of membrane and organelles. Each pellet was
suspended in 75% ethanol containing 50 g l " 1 glucose and
centrifuged at 8000 x g for 5 min at 2 °C (five times) to remove
all free sugars. This fraction was the membrane fraction.
Purified cell walls and membranes were hydrolysed with 3 N
HC1 in sealed tubes at 120°C, for 1 h. The hydrolysates were
dried, resuspended in water and counted for radioactivity. The
cell wall residue was dissolved in 3% (w/w) H 2 SO 4 and
hydrolysed at 120 °C, for 1 h. The hydrolysate was neutralized
with a 15% (v/v) solution of methyl-di-H-octylamine in chloroform (excess of amine was removed by washing with five
changes of chloroform), taken to dryness, resuspended in water
and counted for radioactivity (cellulose).
The lOOOOOxg SN was treated with 60% ethanol (final
concentration) and centrifuged in a J2-21 centrifuge, rotor
head J-20 for 10 min at lOOOOxg. The pellet was washed five
times with 60% ethanol (v/v). The pellet and aliquots of the
lOOOOxg supernatant were counted for radioactivity in order
to determine radioactive soluble polysaccharides and cytosolic
pool, respectively.
Radioactive protein labelling
Six pea seedlings were incubated in water with or without
10 jig ml" 1 BFA for 1 h, at 25°C, in the dark. During the last
30 min of incubation 8 ^Ci of L-[U-14C]proline, 8 ^Ci of L-[UI4
C]leucine or 750 /nCi of L-[35S]methionine were added to the
incubation medium. At the end of the radiolabelling period,
membranes and purified cell walls were isolated. To evaluate
the radioactivity incorporated into cytosolic proteins, the
lOOOOOxg SN was concentrated (Centricon 10, Amicon) and
the proteins were precipitated with 80% acetone at — 20 °C
overnight. Membrane proteins were obtained from membranes
depleted of lipids as described by Dalessandro et al. (1986).
The proteins were precipitated with 80% acetone (final concentration) at — 20 °C overnight. Purified cell walls were resuspended and agitated in phenol: acetic acid:H 2 0 (2/1/1, w/v/v)
at 70°C for 30min (Fry, 1988) to extract non-covalently/
ionically bound cell wall proteins (PAW extract). After
centrifugation (8700 xg, 10 min) the proteins present in the
supernatant were precipitated with 0.1 M ammonium acetate in
methanol over night. The 8000 x g pellet, consisting of cell wall
and covalently bound proteins, was hydrolysed with 3 N H O
in sealed tubes at 120°C, for l h (HC1 hydrolysate). PAW
extract and H O hydrolysate were dried, resuspended in water
and counted for radioactivity.
Cytosolic, membrane and cell wall proteins were analysed by
SDS-PAGE electrophoresis (Laemmli, 1970) on a 13% polyacrylamide gel. Protein bands were stained with Coomassie
brilliant blue R-250. Labelled proteins were detected by
fluorography on Hyperfilm-/3-max (Amersham).
Double radiolabelling of proteins and cell wall polysaccharides
Six pea seedlings were incubated in water with or without
10/xgmr 1 BFA for 1 h, at 25 °C, in the dark. During the last
30 min of incubation, 8 fid of r>[U-14C]glucose and 100 ^Ci
of L-[4,5-3H]leucine were simultaneously added to the medium.
After radiolabelling, the cytosolic pool, soluble polymers,
1927
membrane and cell wall polysaccharide and proteins were
isolated.
Mannan and glucomannan synthesis
The effect of BFA (10 and 50/igml" 1 for 30 min) on the in
vitro synthesis of mannan and glucomannan was studied by
using membrane-bound and digitonin-solubilized enzymes isolated from the third internode of pea seedlings. Enzymic
preparations, incubation procedure and isolation of radioactive
polymers were performed as reported by Piro et al. (1993).
Enzymic assays were performed by using membrane-bound or
digitonin-solubilized enzymes preincubated or not in the
presence of BFA.
Radioactivity counting procedure
Radioactivity counting procedure was performed as described
by Piro et al. (1993), using a 1217 Rack Beta liquid scintillation
spectrometer (LK.B). Counting efficiency was approximately
90%.
Results
All experiments used 2-d-old pea seedlings which were
incubated for short times with and without BFA in the
presence of D - [ U - 1 4 C ] glucose, L-[U-14C]leucine, L - [ U 14
C]proline or both D-[U-14C]glucose and L - [ 4 , 5 - 3 H ]
leucine. Segments (1 cm) of decapitated roots were used
to estimate the incorporation of radioactive sugars and
amino acids into cell wall polysaccharides and proteins.
Incorporation data are expressed as percentages of the
total radioactivity measured in the homogenate of the
root segments.
BFA at 10 and 20 jig m l " 1 inhibited D-[U- 1 4 C]glucose
uptake in pea seedlings by approximately 16% and 50%,
respectively, up to 1 h of incubation (Fig. 1A). A more
pronounced inhibition by BFA was observed in the total
amount of radioactive material found in the homogenates
obtained from decapitated root segments isolated from
BFA-treated seedlings (Fig. IB). The incorporation of
radioactive sugars into matrix polysaccharides was inhibited by BFA (10/^g ml" 1 ) by approximately 17% and
30% after 30 and 60 min, respectively. BFA at 20 jig ml" 1
markedly inhibited the incorporation of radioactive
sugars into matrix polysaccharides from 15 min of incubation (Fig. 1C). At 10 and 20 jig ml" 1 of BFA, the biosynthesis of cellulose, expressed as a percentage of radioactive
glucose incorporated into the cellulose, was drastically
inhibited after 15 min of incubation. BFA at 10 jig ml" 1
inhibited cellulose synthesis more rapidly than matrix
polysaccharides.
To test how BFA affects the synthesis and transport of
cell wall polysaccharides to the wall, pea seedlings were
incubated for 1 h with and without BFA (10 jig ml" 1 )
and after 30 min of incubation, D-[U- U C]glucose was
added in the medium. In these experimental conditions
the inhibitory effect of BFA on the synthesis of cell wall
polysaccharides had already started before the addition
1928
Lanubile et al.
100
Table 1. Effect of BFA on radioactive lOOOOOxg SN and
radioactive polvsaccharides found in membrane and purified
cell wall
(A) D-[U- 14 C]glucose uptake
80-
The subcellular fractions were isolated from 1 cm long root segments
obtained from three pea seedlings incubated in water with or without
lOngml" 1 BFA for 1 h, at 25°C, in the dark. During the last 30min
of incubation, 4 ^Ci of D-[U-'4C]glucose were added in the medium.
Incorporation is expressed as a percentage of the total amount of the
radioactivity measured in the homogenate. The data are from one
representative experiment of three.
GO40 •
20-
Radioactivity
(% of the homogenate)
50 •
(B) homogenate
40 •
lOOOOOxgSN
Membrane
Cell wall
Matrix polysacchandes
Cellulose
Total radioactivity
of the homogenate (kBq)
302010 •
H2O
lO^gml" 1 BFA
73 9
1 8
87 0
1.7
16.6
7.7
9.5
1.8
20.4
20.1
10 '
(C) matrix polysaccharidcs
8
6 •
4
•
2-
10 •
(D) cellulose
8
6
4 '
2 •
15
30
45
I
60
Incubation time (min)
Fig. 1. Effect of BFA on: uptake of D-IU-^Clglucose by pea seedlings
(A), radioactive material present in the homogenate (B), radioactive
sugars incorporated into matrix polysaccharides (C) and cellulose (D)
in 1 cm long pea root segments. 2-day-old pea seedlings (three) were
incubated in water with or without BFA (10 and 2 0 n g m l " 1 ) in the
presence of 4^Ci of D-[U-14C]glucose for 15, 30 and 60 min. At the
end of incubation period, the seedlings were rapidly washed in a
solution of cold glucose and depleted of root cap. Starting from the
root apex, a segment 1 cm long was cut from each root and processed
to analyse the amount of radioactivity present in the homogenate and
incorporated into cell wall polysaccharides. ( • ) Water, (A) BFA
10 fig m l " ' , ( • ) BFA 20 ^g m l " ' . The data are from one representative
experiment of three.
of radioactive tracer. Table 1 shows that the percentage
of radioactive sugars incorporated into cellulose and
matrix polysaccharides in both membranes and cell walls
was inhibited in root segments isolated from BFA-treated
pea seedlings. The inhibitory effect of BFA on the matrix
polysaccharides in membranes and cell walls was 6% and
43%, respectively. Under the same conditions, cellulose
was inhibited by approximately 77%. The lack of any
accumulation of radioactive matrix polysaccharides in
membranes suggested that an inhibition of matrix polysaccharide synthesis occurred before an inhibition of their
transport to the wall.
Pea seedlings were incubated for 1 h with BFA
( l O ^ g m l " 1 ) , and during the last 30 min of incubation,
L-[U-14C]proline or L-[U-14C]leucine was added to the
medium. BFA inhibited the uptake of L-[U-14C]proline
and L-[U-14C]leucine by approximately 60% and 50%,
respectively. A similar inhibition was found for the total
amount of radioactive material present in the homogenates. However, the percentage of incorporation of L-[U14
C]leucine and L-[U-14C]proline into cytosolic, membrane and cell wall proteins was only slightly changed in
the presence of BFA (Table 2). In particular the
percentage of L-[U-14C]leucine and L-[U-14C]proline
incorporated into both non-covalently/ionically bound
(PAW extract) and covalently bound (HC1 hydrolysate)
cell wall proteins was slightly stimulated. The synthesis
and transport of newly synthesized proteins to the cell
wall was only slightly affected by BFA under similar
conditions to those which brought about a strong inhibition of the synthesis of matrix and cellulosic
polysaccharides.
Confirmation that the biosynthesis of cell wall polysaccharides was inhibited by BFA without affecting the
synthesis and transport of cell wall proteins was obtained
when pea root seedlings were incubated for 1 h with BFA
and during the last 30 min D-[U-14C]glucose and
L-[4,5-3H]leucine were added simultaneously to the
incubation medium. The percentage of [ 3 H]material
BFA: cell wall and protein synthesis
4
1929
14
Table 2. Effect of BFA on the incorporation of L-[U-' CJprohne or L-[U- C] leucine into cytosolic, membrane and cell wall proteins
extracted from pea root segments
Cytosolic, membrane and cell wall proteins were isolated from 1 cm long root segments obtained from six pea seedlings incubated in water with or
without 10/igml' 1 BFA for 1 h, at 25 °C, in the dark. During the last 30 min of incubation 8 MCi of L-[U-14C]proune or 8/xCi of L-[U-14C]leucine
were added to the medium. Incorporation is expressed as a percentage of the total amount of radioactivity measured in the homogenate. The data
are from one representative experiment of three.
Proteins
Radioactivity (% of the homogenate)
L-[U-14C]proline
Cytosolic
Membrane
Cell wall
PAW extract
HC1 hydrolysate
Total radioactivity of
the homogenate (kBq)
L-[U-14C]leucine
H2O
10 Mg ml" 1 BFA
H2O
10 y.% ml" 1 BFA
18.4
12.4
17.4
11.8
19.0
11.9
14.3
12.0
1.8
0.2
2.2
0.4
3.1
0.6
3.4
0.8
51.3
14.7
60.3
22.9
present in the cytosolic pool and the percentage of
radioactive leucine incorporated into soluble, membrane
and cell wall proteins was only slightly affected by BFA
(Table 3) but the incorporation of [14C]sugars into cell
wall polysaccharides and into the oligosaccharide sidechains (much less) of cell wall glycoproteins was inhibited.
In addition, the percentage of radioactive polymers found
in membranes (matrix polysaccharides and the oligosaccharide side-chains of glycoproteins and glycolipids) remained almost unchanged in BFA-treated seedlings. The
percentage of radioactive sugars incorporated into both
soluble polysaccharides and glycoproteins which co-precipitated with cytosolic proteins was stimulated by BFA.
The pattern of newly synthesized cytosolic, membrane
and cell wall proteins isolated from BFA-treated and
untreated pea-root seedlings incubated in the presence of
L-[35S]methionine is shown in Fig. 2. BFA does not
change the pattern of proteins in the three fractions. The
proteins extracted from purified cell walls of BFA-treated
and untreated roots remained qualitatively and quantitatively the same. This confirmed that the synthesis and
transport of newly synthesized proteins into the wall were
not changed by BFA. Similar results were obtained when
L-[35S]methionine was substituted from L - [ U - 1 4 C ] leucine
(data not shown).
BFA (10 and 50 fig ml" 1 ) on membrane-bound and
digitonin-solubilized mannan and glucomannan synthase
isolated from the third internode of pea seedlings did not
change the specific activity of the synthases (Table 4)
even when the membrane-bound and the digitoninsolubilized enzymes were pretreated for 30 min with BFA
before the addition of substrates (data not shown).
The inhibition of polysaccharide synthesis produced in
pea-root seedlings by BFA was eliminated after its
removal (Fig. 3). The synthesis of cellulose recovered
rapidly from the BFA effect, reaching 94.5% and 100%
of the control level after 20 and 30 min, respectively. The
recovery of the synthesis of matrix polysaccharides by
Table 3. Effect of BFA on the incorporation of D-[U-l4CJglucose and L-/4,5-3H]leucine into soluble, membrane and cell wall polymers
(proteins and polysaccharides) extracted from pea root segments
1 cm long pea root segments were isolated from six pea seedlings incubated in water with or without lOfig ml" 1 BFA for 1 h, at 25°C, in the dark.
During the last 30 min of incubation 8 ^C\ of r>[U-14C]glucose and 100 ^Ci of L-{4,5-3H]leucine were simultaneously added to the medium.
Incorporation is expressed as a percentage of the total amount of the radioactivity measured in the homogenate. The data are from one
representative experiment of three.
Radioactivity (% of the homogenate)
tO/xgml-'BFA
H2O
14
D - [ U - C ] glucose
Cytosolic pool
Soluble polymers
Membrane
Cell wall
PAW-extract (proteins)
HC1 hydrolysate (matrix polymers)
H 2 SO 4 hydrolysate (cellulose)
Total radioactivity
of the homogenate (kBq)
3
L-{4,5- H] leucine
D-[U-'4C]glucose
L-[4,5-3H] leucine
40.9
I.I
7.2
23.5
3.7
6.0
45 0
1.9
7.5
25 2
4.1
6.1
2.1
6.8
5.9
2.2
0.3
1.7
3.7
3.1
2.2
0.2
31.9
167.0
25.0
82.3
1930
Lanubile et al.
membrane
200.0
97.4
69.0
kDa
—
—
—
46.0
—
30.0
—
cytosol
14.3
H2O
BFA
H2O
BFA
H2O
BFA
Fig. 2. Pattern of cell wall, membrane and cytosolic proteins extracted
from BFA-treated and untreated pea root seedlings. Cell wall, membrane
and cytosolic proteins were isolated from 1 cm long root segments
obtained from 30 pea seedlings incubated in water with or without
l O ^ g m r 1 BFA for 1 h, at 25 °C in the dark. During the last 30min
of incubation 750 ^Ci of L-[35S]methionine were added in the
medium The amount of radioactive proteins on each lane is approximately 250000 dpm. Standard protein molecular-weight markers (kDa)
are on the left.
BFA although slower than that of cellulose was established between 20 and 30 min.
Discussion
The results show that in pea root seedlings BFA rapidly
and markedly inhibits the synthesis of both cellulose and
cell wall matrix polysaccharides without altering the synthesis and transport of newly synthesized proteins into
the walls. Therefore under these experimental conditions
the inhibitory effect of BFA on cell wall polysaccharides
cannot be due to an inhibition of vesicular traffic to the
wall, but to an indirect or direct effect on the activity of
polysaccharide synthase complexes located at the
membranes.
Driouich et al. (1993) working with sycamore suspension-cultured cells showed that, although the newly synthesized cellular proteins in sycamore cells were only
slightly inhibited by BFA, their secretion into the culture
medium was inhibited. The incorporation of [3H]xylose
from UDP-D-[3H]xylose and [3H]fucose into the hemicelluloses extracted from the cell wall of BFA-treated sycamore cells was also inhibited. The close correlation
between the inhibitory effect of BFA on protein secretion
into the culture medium and the reduced amount of
radioactive sugars incorporated into the hemicelluloses
extracted from the walls was taken as proof that BFA
brought about its effects by an inhibition of the secretory
pathway. However, using the same experimental conditions they showed that the incorporation of [ 3 H]mannose,
[3H]xylose from UDP-D-[3H]xylose and [3H]fucose into
the oligosaccharide chains of glycoproteins secreted
within the cell or to the culture medium was differentially
inhibited by BFA. They suggested that the drug acted on
the synthesis and/or processing of N-linked glycans.
However, Driouich et al. (1993) gave no explanation for
the fact that the amount of newly synthesized glycoproteins secreted into the culture medium calculated as
percentage with respect to newly synthesized cellular
glycoproteins remained unchanged or was even stimulated
in BFA-treated sycamore cells compared to that of control
cells. Thus, if the inhibitory effect of BFA was mainly
due to an inhibition of the vesicular traffic, as postulated
by Driouich et al. (1993), it is difficult to explain why
BFA did not alter but even stimulated the secretion of
unglycosylated or incorrectly glycosylated proteins into
the culture medium.
The results of this study show that, in pea root seedlings, cellulose and cell wall matrix polysaccharides are
inhibited by BFA under experimental conditions in which
it is possible to verify that (a) the synthesis and transport
Table 4. Effect of BFA on membrane-bound and digitonin-solubilized mannan and glucomannan synthase isolated from the third
internode of pea seedlings
Mannan- and glucomannan-synthase activity was measured as the amount of either [14C]mannose or [14C]glucose incorporated in the [14C]polymer.
For the mannan-synthase activity the reaction mixture contained: 0 20 nmol of GDP-r>[U-14C]mannose (approximately 1500 Bq), 2.5 nmol of
GDP-D-mannose, BFA (10 or 50^gml~') and membrane enzymic preparation (180^g of protein) or digitonin-solubilized enzymes (50^g of
protein). For the glucomannan-synthase activity the reaction mixture contained: 0.27 nmol of GDP-D-[U-'4C]glucose (approximately 1500 Bq),
1.0 nmol of GDP-D-glucose, 5.0 nmol of GDP-D-mannose, BFA (10 or 50 ^g ml" 1 ) and membrane enzymic preparation (180 ^g of protein) or
digitonin-solubilized enzymes (50^g of protein). Reaction time at 27 °C was 30 min. The data are from one representative experiment of three.
BFA
Specific activity (nmol min" 1 mg" 1 of protein)
Membrane-bound enzyme
50 jig ml
Digitonin-solubilized enzyme
Mannan-synthase
Glucomannan-synthase
Mannan-synthase
Glucomannan-synthase
3.80
3.70
3.75
0.40
0.45
0.38
1.90
2.00
1.85
0.22
0.20
0.24
BFA: cell wall and protein synthesis
IWWIMUWmmatM
0
incubation time (min)
^
1
60
I I
1520
I
30
I
60
recovery time (min)
H2O
BFA 10 |ig ml-1
D-[U-14C] glucose
15 20
30
Recoverytime(mm)
Fig. 3. Recovery of the biosynthesis of cell wall polysacchandes from
pretreatment with BFA in pea root seedlings. 1 cm long pea root
segments were isolated from 3 pea seedlings incubated with or without
10 ^g ml" 1 BFA for 60 min, in the dark at 25 °C. The recovery from
the effect of BFA was studied by transferring seedlings in water for 15,
20, 30, and 60 min and adding 4 ftCi of D-[U-14C]glucose as radioactive
tracer in the last 10 min of incubation as reported in the scheme. (A)
Cellulose, ( • ) matrix polysaccharides. The data are from one
representative experiment of three.
of newly synthesized proteins into the wall was not
affected by the drug (Tables 2, 3); (b) the patterns of
newly synthesized cytosolic, membrane-bound and cell
wall proteins isolated from BFA-treated and control roots
were similar (Fig. 2); (c) the inhibition of cellulose synthesis started at a concentration of BFA which did not
affect the synthesis and transport of cell wall matrix
polysaccharide into the wall. These data show that the
inhibitory effect of BFA on cell wall polysaccharide
synthesis started before any effect on the vesicular traffic
of proteins to the walls. In support of this idea, Kimura
et al. (1993) have demonstrated that the application of
1931
BFA to suspension-cultured rice cells inhibited the synthesis of O-linked saccharide chains (recognized by peanut
lectin (PNA) and Ulex europaeus lectin-I (UEA-I)) of
Golgi membrane glycoprqteins. It was also shown that
both the protein composition of Golgi membrane and the
intracellular transport of a-amylase molecules were not
affected by BFA. It has also been reported that as a
consequence of BFA treatment an altered O- and Nglycosylation occurred in mammalian and plant cells
(Chawla and Hughes, 1991; Collins and Mottet, 1992;
Sampath et al., 1992; Driouich et al., 1993; Gomez and
Chrispeels, 1993).
The cellulose synthase complex of the plasma membrane is not only involved in the synthesis of j3-glucan
chains but is also implicated in their association into
micro- and macro-fibrils. The complex has catalytic subunits for the synthesis of /3-glucan chains and a highly
organized number of structural and regulatory proteins
which bring about the crystallisation of /S-glucan chains
into cellulose I (Northcote, 1991; Delmer and Amor,
1995). It was found that BFA, given simultaneously with
a radioactive tracer, rapidly inhibited cellulose synthesis
(Fig. ID). It was also observed that in vitro BFA did not
change the activity of the membrane-bound and digitoninsolubilized mannan and glucomannan synthase isolated
from the third internode of pea seedlings. In animal
systems also, BFA does not affect the sugar nucleotide
transporters or the glycosyltransferases (Misumi et al.,
1986). All the evidence indicates that the inhibitory effect
of BFA on both cellulose and matrix polysaccharides
formation is not at the catalytic site of the synthases.
Little is known about the structural and functional
organization of polysaccharide synthase complexes in
membranes but it is probable that, similar to the cellulose
synthase complex, different proteins such as donor substances, transferases and acceptor molecules are involved
in the synthesis of cell wall matrix polysaccharides. These
proteins must interact very closely with the phospholipids
of membranes since in vitro membrane-bound polysaccharide synthases lose their activity very rapidly in the
presence of phospholipases (Piro et al., 1993). Since BFA
is known to be a lipophilic molecule which interacts with
membranes, the effect of BFA on the biosynthesis of cell
wall polysaccharides could be caused by an interference
of the drug with the topological organization of the
synthase complexes in the membranes. This effect would
precede the action of the drug at the level of vesicle
transport of proteins to the walls. This would explain the
differential sensitivity to the drug, at the Golgi apparatus,
toward the synthesis of cell wall polysaccharides and the
machinery of vesicular transport of proteins to the wall.
A differential action of BFA on membrane compartments
has been reported in animal and plant cells (Hunziker
et al, 1991; Pelham, 1991; Gomez and Chrispeels, 1993).
The synthesis of glycosaminoglycan chains occurs in
1932
Lanubile et al.
most eukaryotic cells (Hardingham and Fosang, 1992). In
condrocytes of chondrosarcoma cells from the swarm rat,
the disruption of vesicular traffic by BFA rapidly inhibited
the synthesis of chondroitin sulphate, the major structural
components of proteoglycan aggrecan. However, the hyaluronan synthase complex, at the plasma membrane, was
much more stable and hyaluronan synthesis continued at
the same rate up to 8 h. The recovery of chondroitin
sulphate synthesis from the action of BFA after its removal
occurred in the presence of cycloheximide so that its
synthesis probably reassembled from previously existing
proteins (Calabro and Hascall, 1994a, b). These results, in
contrast to our results, indicated that BFA had no effect
on the glycosyltransferases involved in the chondroitin
sulphate and hyaluronan synthesis. It must be noted,
however, that the morphology and function of the Golgi
apparatus differ between plant and animal cells. It is also
established, as has already been indicated, that the action
of BFA varied not only with the concentration of the drug
applied to the tissue but even between different species of
plants. Differences between the action of BFA on plant
and animal cells might therefore be expected. It is possible
that a more complex organization of polysaccharide synthases including the glycosyltransferases, is present on plant
membranes compared to that of animal cells. No polysaccharide synthases have been purified and characterized
from higher plants whereas several glycosyltransferases
have been purified and characterized in animal systems
(Paulson and Colley, 1989; Kleene and Berger, 1993).
The rapid inhibition of cellulose and matrix polysaccharide synthesis by BFA in pea-root seedlings is paralleled
by a rapid recovery of the inhibitory effect when BFA is
removed so that BFA probably interacts non-covalently
with molecules such as lipids, proteins or both in an
hydrophobic environment. This would explain why the
effects of BFA on membranes are not only multi-targeted
(Pelham, 1991; Klausner et al., 1992), but also cell-type
specific (Hunziker et al., 1991; Satiat-Jeunemaitre and
Hawes, 1994). The many different responses to BFA both
biochemically and morphologically, may depend on the
unique lipid and protein composition of membrane compartments in plant and animal cells.
Acknowledgements
The authors are grateful to Professor DH Northcote (Cambridge
University) for a critical reading of the manuscript. We also
thank Professor A Ballio (Rome University) for the suggestion
to study BFA on plant cells. This research was supported by
MURST 40%.
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