Plant Cell Physiol. 41(7): 840–849 (2000)
JSPP © 2000
Mechanism of the Chilling-Induced Decrease in Proton Pumping across the
Tonoplast of Rice Cells
Kunihiro Kasamo 1, 3, Mineo Yamaguchi 1 and Yoshiyuki Nakamura 2
1
2
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
Chugoku National Agricultural Experiment Station, Fukuyama, Hiroshima, 721-8514 Japan
;
pressed by chilling (Yoshida and Matsuura-Endo 1991).
Further studies are required to clarify the mechanism responsible for the decrease in ATP-generated proton pumping across
the tonoplast by chilling.
The decrease in the activity of the tonoplast H+-ATPase by
chilling is reportedly due to selective release of peripheral subunits of ATPase in the tonoplast (Moriyama and Nelson 1989,
Parry et al. 1989, Matsuura-Endo et al. 1992). On the other
hand, tonoplast H+- ATPase activity (Yamanishi and Kasamo
1993) and proton pumping across proteoliposomes (Kasamo
and Yamanishi 1991) are closely related to the composition of
the membrane phospholipids. Thus, there remains the possibility that the decrease in proton pumping across tonoplast and that
in H+-ATPase activity by chilling are due to the modulation of
lipids surrounding H+-ATPase molecules in the tonoplast.
We tried the reconstitution of chimera proteoliposomes
with H+-ATPase and membrane lipids from a different variety
or plant species. This system should help to clarify the molecular relation between the membrane proteins and membrane lipids. By using these chimera proteoliposomes, we elucidated the
mechanism for the chilling-induced decrease in ATP-generated
proton pumping across the tonoplast.
The local fluidity in the core hydrophobic region and the
surface hydrophilic region of the lipid bilayer of the tonoplast
vesicles or proteoliposomes was individually estimated using
fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene
(DPH) and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene
(TMA-DPH), respectively (Benedetti et al. 1989), and using
electron spin resonance (ESR) spectroscopy of 16- and 5-doxyl
stearic acid (DOSA), respectively (Jost et al. 1971). We further
examined the relation between the local membrane fluidity and
the chilling.
The ATP-generated proton pumping across tonoplast
vesicles from chilling-sensitive Boro rice (Oryza sativa L.
var. Boro) cultured cells was markedly decreased by chilling at 5C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer
was measured by steady-state fluorescence depolarization
of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium
1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer
of the tonoplast vesicles decreased by chilling. The fluidity
of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast
H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro
cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to
the decrease in the fluidity of the surface region of the lipid
bilayer of the tonoplast, which is caused by the changes in
glycolipids.
Key words: Chilling—Chimera proteoliposomes—Glycolipids—Membrane fluidity—Oryza sativa L.—Tonoplast H+ATPase.
Abbreviations: DGDG, digalactosyldiacylglycerol; DOC, deoxycholate; DOSA, doxyl stearic acid; DPH, 1,6-diphenyl-1,3,5-hexatriene;
ESR, electron spin resonance; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl hydrazone; n-OG, n-octylglucoside; TMA-DPH, trimethylammonium DPH.
Materials and Methods
Introduction
Plant material
Chilling-sensitive rice cells (Oryza sativa L. var. Boro) and chilling-insensitive rice cells (Oryza sativa L. var. Nipponbare) were selected from rice cultured cells reported previously (Kasamo 1988,
Kasamo et al. 1992).
The vacuole is the cellular organelle that responds initially to chilling stress in higher plants. The collapse of the vacuole by chilling induces plant cell damage (Yoshida et al. 1979).
The activity of H+-ATPase, which is localized in the tonoplast,
markedly decreased with increasing duration at a low temperature (Kasamo 1988, Yoshida et al. 1989). ATP-generated proton pumping across tonoplast vesicles was also markedly sup3
Isolation of tonoplast vesicles
Tonoplast vesicles were isolated according to the method of
Kasamo and Yamanishi (1991). In brief, approximately 40 g FW of
cultured rice cells with or without chilling were homogenized with a
Corresponding author: E-mail, [email protected]; Fax, +81-(0)86-434-1221.
840
Chilling-induced inhibition of proton pumping
homogenizer after grinding in a chilled mortar and pestle in a 100 ml
of grinding medium containing 0.25 M mannitol, 25 mM HEPES-Tris
(pH 7.5), 2 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,Ntetraacetic acid (EGTA), 1 mM dithiothreitol (DTT) and 0.1% bovine
serum albumin (BSA). The homogenate was centrifuged at 1,500 g
for 10 min. The supernatant was centrifuged at 10,000 g for 30 min.
The resultant supernatant was further centrifuged at 80,000 g for 30
min to obtain the microsomal pellet. The pellet was subsequently suspended in 4 ml of suspension buffer containing 0.25 M mannitol, 10
mM MES-Tris (pH 7.3), 1 mM EGTA and 1 mM DTT. Four ml of the
suspension was layered over a step gradient containing 4.5 ml of 6%
dextran T-70 (w/v) (Pharmacia, Sweden) in the suspension buffer. The
step gradients were centrifuged at 105,000 g for 90 min in a Beckman SW 40 rotor. The interface between 0 and 6% dextran was pipetted, diluted five-fold with the suspension buffer and centrifuged at
140,000 g for 40 min. The resulting pellet, enriched with tonoplast
vesicles, was resuspended in the suspension buffer containing 20% (v/
v) glycerol.
Solubilization of H+-ATPase from tonoplast
A two-step solubilization procedure using deoxycholate (DOC)
(Wako Chemicals, Japan) and n-octylglucoside (n-OG) (Dojin Kagaku Co., Japan) was used to solubilize ATPase from the tonoplast, as
described previously (Kasamo et al. 1991). Briefly, in the first step,
DOC from a 10% stock solution was added dropwise into the tonoplast-enriched membranes (1 mg protein ml–1) in a solubilization buffer containing 1 mM EDTA, 1 mM DTT and 10 mM Tris-MES (pH
7.5) with stirring on ice to make a final DOC concentration of 0.1%.
After 10 min of stirring on ice, the mixture was centrifuged at 140,000
g for 1 h in a Beckman Type 65 rotor. The solubilization buffer containing 45% (v/v) glycerol (solubilization buffer B) was added to the
pellet to give a final protein concentration of approximately 2 mg ml–1.
Most of the bafilomycin-sensitive ATPase remained in the DOC-pellet.
In the second step, n-OG from a 300 mM stock solution was added to
the resuspended DOC-pellet to make a final concentration of 30 mM.
After 10 min of stirring and centrifugation at 140,000 g for 30 min,
most of the ATPase activity was found in the supernatant. The solubilized fractions were diluted five-fold with 1 mM EGTA-Tris (pH 7.3)
and centrifuged at 120,000 g for 4 h to collect the solubilized tonoplast ATPase. The pellet was suspended in solubilization buffer B to
approximately 1 mg protein ml–1.
Lipid extraction and separation
Total lipids were extracted from the tonoplast preparations of
chilled or nonchilled rice cells by the method of Blight and Dyer
(1959). The total lipid extract was partitioned into neutral lipids, glycolipids and phospholipids on silica Sep-Pak cartridges (Waters,
U.S.A.) (Hamilton and Comai 1984, Lynch and Steponkus 1987).
Briefly, the total lipid extracts were evaporated to near dryness under a
stream of nitrogen gas, dissolved in 2 ml of chloroform: acetic acid
(100 : 1, v/v), and then transferred to the Sep-Pak cartridge attached to
a 10 ml disposable syringe. After the sample had entered the cartridge,
2 ml of chloroform: acetic acid (100 : 1, v/v) was used to wash the residual lipids from the original container. Ten ml of the same solvent
was used to elute the neutral lipids from the column. The glycolipids
were eluted next with 10 ml of acetone followed by 10 ml of acetone :
acetic acid (100 : 1, v/v). The phospholipids were eluted last with 10
ml of methanol : chloroform : water (100 : 50 : 40, v/v/v) and recovered from this fraction by the addition of 1 ml chloroform and 2 ml
water to facilitate the phase separation. Digalactosyldiacylglycerol
(DGDG) was washed with 10 ml acetone, followed by 15 ml acetone :
acetic acid (100 : 1, v/v) after elution of neutral lipids. All of the
eluted lipid fractions were concentrated to near dryness under a stream
841
of nitrogen gas. The concentrated neutral lipids were dissolved and
stored in either hexane or chloroform. The glycolipids were dissolved
and stored in chloroform : methanol (1 : 1, v/v). The phospholipids
were dissolved and stored in either chloroform : acetic acid (100 : 1,
v/v) or chloroform : methanol (19 : 1, v/v). All the samples were
stored at 80C prior to the analysis. Individual lipids were identified
by co-chromatography with authentic standards and the use of specific
spray reagents (Kates 1972). Lipid sugar content was quantified by the
method of Roughan and Batt (1968). The lipid phosphorous content
was determined according to the method of Rouser et al. (1970).
Reconstitution of tonoplast H+-ATPase into liposomes
The reconstitution method of Kasamo et al. (1991) was slightly
modified. Fifty to 200 g of solubilized ATPase in solubilization buffer B and 10 mg of sonicated asolectin (purified phospholipid mixture)
or individual lipids and/or combined lipids extracted from the tonoplast in 0.1 ml of reconstitution buffer consisting of 10 mM MES-Tris
(pH 7.2), 100 mM KCl and 1 mM EDTA were mixed in 1.2 ml total
volume of reconstitution buffer. A 72 l aliquot of 10% (w/v) DOC
was then added to this cloudy suspension for a final concentration of
0.6%, which produced a clear suspension. This clear mixture of detergent-protein-lipid was applied to the top of a PD-10 column (Pharmacia, Uppsala, Sweden) which had been equilibrated with a washing
buffer containing 10 mM MES-Tris (pH 7.2), 100 mM KCl and 0.1
mM EDTA. The column was eluted with washing buffer at a flow rate
of 1 ml min–1. Cloudy void volume fractions were pooled, diluted fivefold with washing buffer and centrifuged at 100,000 g for 1 h to precipitate the reconstituted proteoliposomes. The pellet was suspended in
washing buffer at a final concentration of ca. 2 mg protein ml–1 for the
assay of proton pumping.
Fluorescence quenching
For assay of the transport of protons under standard conditions,
vesicles (100–150 g protein) or reconstituted proteoliposomes (20–30
g protein) were mixed in a fluorescence cuvette containing 2.0 ml of
reaction medium (0.25 M sucrose, 100 mM KCl, 10 mM MES-Tris,
pH 6.5, 1 M quinacrine) at room temperature. The reaction was initiated by the addition of 3 mM ATP-MgSO4. Proton transport was followed by monitoring the decrease in quinacrine fluorescence with a
spectrofluorometer (model RF-5000, Shimadzu, Kyoto, Japan) at excitation and emission wavelengths of 425 and 500 nm, respectively. The
maximum pH gradient was assessed by carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)-inducible reversion of the
quenching of quinacrine fluorescence.
Fluorescence depolarization measurement
Fluidity of core hydrophobic and surface hydrophilic regions of
the lipid bilayer of the tonoplast or proteoliposomes was estimated
from steady-state fluorescence polarization measurements using DPH
and its cationic derivatives, TMA-DPH, respectively (Benedetti et al.
1989). Tonoplast vesicles equivalent to 100 g of protein were incubated in 2 ml of 1 mM EGTA/Tris (pH 7.5) containing 1 M DPH or
TMA-DPH. The steady-state fluorescence depolarization of DPH and
TMA-DPH was measured using a fluorescence depolarization spectrophotometer (FS-501A, Otsuka Electronics, Japan) equipped with a
temperature-controlled cuvette holder with excitation and emission
wavelengths at 350 nm and 425 nm, respectively. The temperature during the measurement was increased stepwise by 5C from 5C to
50C. The data were expressed as the fluorescence anisotropy, r, and
the anisotropy parameter ((r0/r)1)–1 was calculated, where the value
of r0, the maximal limiting anisotropy of DPH, was presumed to be
0.362 (Shinitzky and Barenholz 1974).
842
Chilling-induced inhibition of proton pumping
Table 1
Effects of chilling on ATPase activity in various fractions
ATPase activity (mmol Pi (mg protein)–1 min–1)
Nipponbare
Boro
Fractions
Chilling
Chilling
Chilling
Chilling
Tonoplast
Solubilized enzyme
1.320.12
2.230.31
1.170.09
2.280.28
1.050.11
1.780.15
0.440.05
1.310.10
Proteoliposomes
1.190.11
1.010.10
1.160.10
1.030.18
All fractions were prepared from Nipponbare and Boro cells with and without chilling as detailed in Materials and Methods. Values show the average of three independent experimentsSE.
ESR measurement
The membrane fluidity was also assessed by ESR of spin-labeled derivatives of stearic acid with doxyl nitroxide embedded at a
different position along the acyl chain (Jost et al. 1971). 16- and 5DOSA were used for the measurement of membrane fluidity in the
core and surface regions of the lipid bilayer of the asolectin liposomes
containing various concentrations of glucocerebroside, respectively.
Ten g of these DOSAs in methanol was pipetted, and the methanol
was removed under a stream of nitrogen gas to form a fatty acid film.
A suspension of asolectin liposomes equivalent of 100 g was added
to the film and incubated at 5C overnight. ESR measurements were
performed using an ESR spectrometer (JES-RE 3X, JEOL) having a
rectangular cavity with the temperature controlled by a nitrogen gas
stream. ESR spectra were obtained in the X-band region at a microwave power of 10 mW and field modulation frequency of 100 Khz.
The scanning range for recording the spectra was 100 G.
Protein determination
Protein content was determined according to Bradford (1976) or
Peterson (1977) using BSA as the standard.
Results
Effects of chilling on the activity of H+-ATPase in various
preparative fractions
The specific activity of ATPase in tonoplast vesicles from
Boro cells was greatly reduced by chilling at 5C for 3 d. On
the other hand, the activity of ATPase in the enzyme fraction
solubilized from tonoplast vesicles of Boro cells and in proteoliposomes reconstituted with asolectin liposomes hardly showed
any decrease due to chilling (Table 1). Table 2 shows the recovery of ATPase activity and protein solubilized from tonoplast
vesicles of chilled or nonchilled Boro cells. Chilled and nonchilled Boro cells had almost the same level of total protein and
total ATPase activity in the solubilized enzyme fraction. These
data imply that ATPase molecules in vacuolar membranes are
not directly inactivated by chilling for 3 d.
Effect of chilling on proton pumping across tonoplast vesicles
from Nipponbare and Boro cells
Fig. 1 shows the proton pumping across the tonoplast vesicles from Nipponbare and Boro cells which had been chilled at
5C for 3 d. The proton pumping across the tonoplast vesicles
from Boro cells was suppressed by chilling, but the decrease in
chilled Nipponbare cells was hardly observed. Tonoplast vesicles from Boro cells were slightly leaky to protons after chilling for 3 d. The leakage was much less than the reduction in
the proton pumping by chilling (data not shown). We further
examined whether chilling directly affected the H+-ATPase
molecules or lipids resulting in decrease proton pumping.
Table 2 Recovery of total protein and total ATPase activity in tonoplast vesicles and the
enzyme fraction solubilized form tonoplast vesicles of chilled or nonchilled Boro cells
Fractions
Tonoplast vesicles
Total protein (mg)
Specific activity (mol Pi (mg protein)–1 min–1)
Total activity (mol Pi min–1)
Enzyme fraction
Total protein (mg)
Specific activity (mol Pi (mg protein)–1 min–1)
Total activity (mol Pi min–1)
Boro
Chilling
Chilling
1.560.25
1.050.11
1.640.17
1.370.20
0.440.05
0.600.11
0.220.02
1.780.15
0.390.08
0.250.03
1.310.10
0.330.05
Tonoplast vesicles were prepared from 20 g of Boro cells. The enzyme fractions were prepared from tonoplast vesicles by the solubilization with 30 mM OG after treatment with 0.1% DOC. Values show the average
of three independent experimentsSE.
Chilling-induced inhibition of proton pumping
843
Fig. 1 Effect of chilling on proton pumping across tonoplast vesicles from chilling-insensitive and chilling-sensitive rice cells. Tonoplast vesicles were isolated from chilling-insensitive (Oryza sativa L. var. Nipponbare) and chilling-sensitive (Oryza sativa L. var. Boro) rice cultured cells
after chilling at 5C for 3 d. Proton pumping across tonoplast vesicles was monitored by fluorescence quenching of quinacrine at room temperature. The reaction mixture for proton pumping consisted of 0.25 M sorbitol, 100 mM KCl, 10 mM MES-Tris (pH 7.3), 1 M quinacrine and 100
g membrane protein in a final volume of 2 ml. Proton transport was initiated by ATP-MgCl2 (arrows) at a final concentration of 3 mM. The proton gradient collapsed by the addition of 2.5 M FCCP (arrows).
Direct interaction between chilling and H+-ATPase
In order to clarify the direct interaction between chilling
and H+-ATPase, we reconstituted proteoliposomes with asolectin (phospholipid mixture) liposomes and H+-ATPase solubilized from the tonoplast vesicles of Nipponbare or Boro cells
which had been chilled for 3 d (Fig. 2). The proton pumping
across the proteoliposomes with H+-ATPase solubilized from
chilled Boro cells was similar to that from nonchilled Boro
cells as shown in the Nipponbare cells. These findings show
that the reduction in proton pumping due to chilling was not
due to the contribution of the direct interaction between H+-ATPase molecules and chilling.
Direct interaction between chilling and tonoplast lipids
We designated the proteoliposomes reconstituted with a
membrane protein and lipid(s) from different varieties or species as chimera proteoliposomes. Tonoplast lipids were individually extracted for phospholipids, glycolipids and neutral lipids, and used for reconstitution. In order to clarify the direct
interaction between chilling and lipids, we reconstituted chimera proteoliposomes with H+-ATPase solubilized from the tonoplast of Nipponbare cells without chilling and individual or
combined lipids extracted from the tonoplast of Boro cells with
or without chilling at 5C for 3 d. Proton pumping across the
chimera proteoliposomes with tonoplast H+-ATPase and tonoplast phopsholipids hardly decreased due to chilling (Fig. 3a).
However, in the chimera proteoliposomes with tonoplast H+ATPase and the tonoplast lipid mixture containing glycolipids
in addition to the phospholipids, the proton pumping decreased
by chilling (Fig. 3b) as in the tonoplast vesicles from Boro cells
(Fig. 1). Collapse of the proton gradient from the chimera proteoliposomes due to FCCP slowly appeared, that is, the permeability of protons across the proteoliposomes was markedly de-
creased by addition of the tonoplast glycolipids from chilled
Boro cells (Fig. 3b). With the further addition of tonoplast neutral lipids, the proton pumping decreased by chilling and the
stability of the proton permeability recovered (Fig. 3c). These
data show that the chilling-induced reduction of proton pumping appeared only in the presence of the tonoplast glycolipids
which were extracted from chilled Boro cells. The glycolipid
fraction from nonchilled Boro cells contained ca. 18% glucocerebroside, 29% acylsteryl glucoside, 18% steryl glucoside
and 35% digalactosyl diglyceride (data not shown).
The relation between chilling and tonoplast membrane fluidity
We further focused on the relation between membrane fluidity and chilling. Cooke et al. (1994) showed that there was no
significant relation between plasma membrane fluidity and
ATP-generated proton pumping using four kinds of sterols.
However, they used plasma membrane and measured the bulk
membrane fluidity of the membrane, and not the local one.
DPH and TMA-DPH are believed to be localized in the
core and surface regions of the lipid bilayer due to their hydrophobicity and thus can be used as a probe for local fluidity in
the core and surface regions of the membrane lipid bilayer, respectively (Benedetti et al. 1989). In both Nipponbare and
Boro cells, the polarization of the surface region of the lipid bilayer of the tonoplast is higher than that of the core region,
meaning that the membrane fluidity of the surface region of the
tonoplast of rice cells is more rigid than that of the core region
(Fig. 4). Fig. 4 also shows the effect of chilling on the polarization of the surface hydrophilic region and core hydrophobic region of the tonoplast vesicles from the Nipponbare and Boro
rice cells. In both Boro and Nipponbare, chilling increased the
fluidity in the core hydrophobic region. Chilling also increased
the polarization of the surface region of the tonoplast vesicles
844
Chilling-induced inhibition of proton pumping
Fig. 2 Proton pumping across proteoliposomes reconstituted with asolectin liposomes and H+-ATPase solubilized from the tonoplast of chilled
and nonchilled Nipponbare and Boro rice cells. Proteoliposomes were formed by removing DOC from a mixture of 50 g of H+-ATPase solubilized from tonoplast of chilled or nonchilled Nipponbare and Boro rice cultured cells, 10 mg purified asolectin and 0.6% DOC. Proteoliposomes
equivalent to 30 g protein were used to measure proton pumping. Proton pumping was carried out as described in Fig. 1.
from the Boro rice cells. Thus, the fluidity of the surface region of the lipid bilayer of the tonoplast of Boro rice cells was
decreased by chilling. Why does chilling induce the reduction
in membrane fluidity of the surface region of the lipid bilayer?
The relation between membrane fluidity and glycolipids
The reduction in the proton pumping across the tonoplast
by chilling may be related to glycolipids as shown in Fig. 3b.
The tonoplast contains a large amount of glycolipids, especially
cerebroside accounted for 16 mol% of the total tonoplast lipids
(data not shown) as shown by the mung bean tonoplast (Yoshida and Uemura 1986). Galactocerebroside was little in the tonoplast vesicles (data not shown). A large amount of individual
glycolipids is needed to make reconstituted vesicles. Thus, we
used commercially available human glucocerebroside (Sigma)
as the glycolipids to make the proteoliposomes in the following experiments. We measured the fluorescence anisotropy of
the surface region and core region of asolectin liposomes containing various concentrations of cerebroside (Fig. 5). The concentration dependency of cerebroside on the anisotropy of the
core region was hardly observed, but the anisotropy of the surface region increased with increasing concentration of cerebroside. This result shows that the fluidity of the surface region of
the asolectin liposomes decreased with increasing concentration of cerebroside.
The membrane fluidity was also assessed by ESR of the
spin-labeled derivatives of stearic acid with doxyl nitroxide
embedded at a different position along the acyl chain (Fig. 6).
Chilling-induced inhibition of proton pumping
845
Fig. 3 Proton pumping across chimera proteoliposomes reconstituted with tonoplast H+-ATPase from the nonchilled Nipponbare cells and lipids from the tonoplast of chilled and nonchilled Boro cells. Chimera proteoliposomes were formed by removing DOC from a mixture of 100 g of
tonoplast H+-ATPase of nonchilled Nipponbare cells, 10 mg individual or combined lipids from chilled or nonchilled Boro cells and 0.6% DOC.
Total lipids extracted from Boro cells were partitioned into neutral lipids, glycolipids and phospholipids on a silica Sep-Pak cartridge. (a) H +ATPase-phospholipids (10 mg), (b) H+-ATPase-phospholipids (8 mg) plus glycolipids (2 mg), (c) H+-ATPase-phospholipids (6 mg) plus glycolipids (2 mg) and neutral lipids (2 mg). Proteoliposomes equivalent to 25 g protein were used to assay proton pumping.
We used 16- and 5-DOSA for measurement of the membrane
fluidity in the core hydrophobic and surface hydrophilic region
of the liposomes, respectively. The order parameter of 16DOSA remained almost constant value, whereas the order parameter for 5-DOSA increased with increasing concentration of
cerebroside. These data, like the fluorescence anisotropy data,
showed that the surface region of the lipid bilayer of the asolectin liposomes is more rigid than the core region and the fluidity of the surface region decreased with increasing concentration of cerebroside. ESR measurements previously showed that
chilling stress leads to an increase in the cell rigidity in roots of
coffee seedlings (Alonso et al. 1997).
The relation between glycolipids and proton pumping
Fig. 7 shows the effect of cerebroside concentration on
proton pumping across proteoliposomes with H+-ATPase. Pro-
ton pumping, above 5%, decreased with increasing concentration of cerebroside. Taken together, these results imply that the
reduction in proton pumping across the tonoplast by chilling is
due to the decrease in fluidity of the surface hydrophilic region
of the lipid bilayer of the tonoplast caused by cerebroside.
Discussion
Experiments using chimera proteoliposomes demonstrated that the reduction in proton pumping across tonoplast vesicles by chilling is due to the modulation of the interaction between H+-ATPase and the tonoplast lipids, which is caused by
glycolipids.
The tonoplast contains a large amount of glycolipids such
as glucocerebroside and acylsteryl glucoside. Glycolipids appear
to serve four general functions in membranes: stabilization,
846
Chilling-induced inhibition of proton pumping
Fig. 4 Effect of chilling on polarization of surface region and core region of lipid bilayer of tonoplast vesicles from chilled and nonchilled Nipponbare and Boro rice cells. Tonoplast vesicles equivalent to 100 g protein were incubated in 2 ml of 1 mM EGTA-Tris (pH 7.5) containing 1
M DPH or TMA-DPH. Polarization of core hydrophobic and surface hydrophilic regions on the lipid bilayer of the tonoplast was estimated by
using DPH and TMA-DPH, respectively. Temperature during the measurement was increased stepwise by 5C from 5C to 50C.
Fig. 5 Fluorescence anisotropy of surface region and core region of lipid bilayer of asolectin liposomes containing various concentrations of
glycolipids. The data are shown as the fluorescence anisotropy. Various concentrations of glucocerebrosides were used as the glycolipids.
shape determination, recognition and ion binding (Guratolo
1987). Cerebroside, a glycolipid, is the major lipid component of
the tonoplast (Yoshida and Uemura 1986, Travernier et al. 1993)
and the plasma membrane (Rochester et al. 1987, Sandstrom and
Cleland 1989) of several plant species. Cerebroside in plants
consists primarily of 8-unsaturated sphingoid bases, 2-hydroxyl
Chilling-induced inhibition of proton pumping
847
Fig. 6 ESR spectra of 5- and 16-DOSA embedded in the lipid bilayer of asolectin liposomes containing various concentrations of glycolipids.
16- and 5-DOSA were used for the measurement of membrane fluidity in the core and surface regions of the lipid bilayer of the asolectin liposomes containing various concentrations of glucocerebrosides, respectively. The values of the order parameter given at the right end of the spectra were calculated using the distance between the peaks indicated by and that between the peaks indicated by (Hubbell and McConnell 1971).
fatty acids, and glucose. Molecular species (Cahoon and Lynch
1991, Imai et al. 1997) and the physicochemical behavior (Norberg et al. 1996) of this lipid have been investigated. This lipid
class is generally considered to physically stabilize the membranes and reduce ion permeability in various animal cells (Curatolo 1987, Karlsson 1987) and may be important in altering the
cryostability of rye and oat membranes (Lynch and Steponkus
1987, Steponkus and Lynch 1989, Uemura and Steponkus 1994).
Cerebroside facilitates the formation of a hexagonal structure in
the presence of sterol (Webb et al. 1997). The tonoplast contains
galactolipids of plastidic origin such as DGDG (Haschke et al.
1990). We could exclude DGDG from the glycolipid fraction.
Glycolipids with or without DGDG decreased the H+-ATPase activity in the presence of phospholipids (Table 3). Since H+-ATPase activity is coupled with proton pumping (Kasamo and Yamanishi 1991) DGDG would not be related to the decrease in
proton pumping.
Proton permeability decreased by addition of glycolipids
and it recovered upon the further addition of neutral lipids (Fig.
3b, c). Previously, we showed that proton pumping across the
asolectin proteoliposome with H+-ATPase containing more
Table 3 Effect of tonoplast glycolipids with or without
DGDG on phospholipid-activated H+-ATPase
Lipids added (25 nmol/assay)
Asolectin
Asolectin glycolipids
Asolectin glycolipids (DGDG)
Fig. 7 Effect of glycolipids on proton pumping across proteoliposomes with H+-ATPase. Glucocerebroside was used as the glycolipids.
Proton pumping and reconstitution were carried out as described in
Fig. 1 and Fig. 2, respectively.
ATPase activity (mol Pi
(mg protein)–1 min–1)
5.41
2.33
2.29
Glycolipids were extracted from tonoplast vesicles by Sep-Pak column.
DGDG was extracted from glycolipids with acetone-acetone acetic
acid (Materials and Methods). Values show the average of duplicate
samples.
848
Chilling-induced inhibition of proton pumping
Table 4
Lipid composition of the tonoplast of rice cells
Nipponbare
Lipids
Chilling
–1
Phospholipids (mmol mg protein )
–1
Glycolipids (mmol mg protein )
0.73
a
0.34 (0.47)
Boro
Chilling
Chilling
Chilling
0.86
0.82
0.93
0.22 (0.26)
0.41 (0.50)
0.33 (0.35)
Total lipids were extracted from the tonoplast of chilled or nonchilled Nipponbare and Boro cells according
to the procedure of Blight and Dyer (1959). The total lipid extracts were separated into neutral lipid, glycolipid and phospholipid fractions using a Sep-Pak silica cartridge. Values show the average of two independent
experiments.
a
The values in parentheses are the molar ratios of glycolipid to phospholipid.
than 5% cerebroside was weaker than that from asolectin alone,
and that cholesterol suppressed proton leakage from proteoliposomes (Yamanishi and Kasamo 1994). We also showed that
purified H+-ATPase was not activated by the exogenous addition of cerebroside (Yamanishi and Kasamo 1993). Because the
glycolipids possess a large polar head group, the hydrocarbon
chains of the glycolipids are obliguely stacked (Gennis 1989).
Thus, glycolipids responsible for the sensitivity to chilling may
induce a conformational change in the H+-ATPase molecules,
resulting in a decrease in proton translocation.
An interesting question is whether the quantitative and
qualitative modulation of these lipid classes is triggered by
chilling. Table 4 shows that the chilling did not increase the absolute quantity of glycolipids, but rather decreased it. However, chilling decreased the relative amount of glycolipids in the
tonoplast from Boro cells less than that from Nipponbare cells.
From these results, in addition to a question of quantity, it does
not deny the following possibilities: (1) chilling may make a
local cluster causing phase separation of the glycolipids near
the H+-ATPase molecules; (2) chilling may induce a certain
molecular species of glycolipid because plants contain trihydroxylcerebroside, and it reduced the membrane fluidity of the
liposomes (data not shown). The variety and structure of plant
sphingoid bases are more complicated than those in animal tissues. Experiments using reconstituted proteoliposomes with
tonoplast glycolipids should be needed. More detailed examinations are in progress.
In summary, the chilling-induced decrease in proton
pumping across the tonoplast is due to the decrease in the fluidity of the hydrophilic surface region of the lipid bilayer of the
tonoplast, which is caused by glycolipids, such as glucocerebroside and acylsteryl glucoside.
Acknowledgments
We would like to thank to Dr. Ohta of Keio University for carrying out the ESR spectrometry. This study was supported in part by
grants from Special Coordination Fund of the Science and Technology
Agency of the Japanese Government, and Grant-in-Aid for Scientific
Research (No. 11440237) from the Ministry of Education, Science,
Sports and Culture of Japan to K.K.
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(Received October 25, 1999; Accepted April 18, 2000)