From the
The electrical activity of a single channel of 525 ± 12
picosiemens in 150 mM KCl was measured after fusion of the
inner envelope membrane of the chloroplast with planar lipid bilayers.
The reversal potentials measured in KCl gradients indicate that this
channel is weakly anion selective
( P
The exchange of solutes between the stroma of the chloroplast
and the cytoplasm proceeds through the envelope made of the inner and
the outer membrane. The outer envelope membrane of intact chloroplasts
has been shown to be freely permeable to molecules of several kDa
(1) . A pore-like channel has been identified in the outer
envelope membrane of spinach chloroplast after detergent-solubilized
membranes were fused with planar lipid bilayers. Its conductance of 720
pS
The inner envelope membrane of chloroplasts is much more selective;
it contains a H
We describe here the molecular properties of a new porin-like
channel that was detected after fusion of the inner envelope membrane
of chloroplasts with a planar lipid bilayer.
The amplitude of the single-channel current was determined from
total amplitude histograms fitted with Gaussian functions. The open
probability of the fully open state ( P
An aliquot of the inner
envelope (5-10 µg of proteins) was sonicated for 20 s and
added to the cis chamber under continuous stirring. Fusion of vesicles
required a large osmotic gradient across the bilayer (cis side: 150
mM KCl, 5 mM CaCl
The conductivity of the
experimental salt solution was measured using a Philips digital
conductivity meter.
In the text, data are expressed as mean ±
S.E. with the number of experiments in brackets. Statistical
significance refers to a Student's t test ( p < 0.05).
Dextran sulfate (8 kDa) (Sigma) was added to both compartments under
continuous stirring for 1 min. Two concentrations were used: 6.3 and 31
µM (final concentrations). At each dextran concentration,
various voltages were applied to determine the channel conductance and
the open probability.
The ratio of the permeability coefficient of
chloride ions to that of potassium ions is calculated from the reversal
potential using the Goldman-Hodgkin-Katz equation. The reversal
potentials measured in three different cis/trans gradients of 450/150
mM KCl, 150/400 mM KCl, and 1000/150 mM KCl
are +5.6 ± 1.6 mV ( n = 4),
We have incorporated a large conductance (525 pS) channel
from the inner envelope membrane of the chloroplast in a planar lipid
bilayer. To our knowledge, it is the first time that a porin is
characterized in the inner envelope of the chloroplast. Like other
porin found in the outer membrane of bacteria, chloroplasts, and
mitochondria, the 525-pS channel has a large conductance, which
increases linearly with salt concentrations (Fig. 4). Moreover,
it is weakly anion selective (Fig. 3). Despite these two
similarities, the 525-pS porin is different from that found in the
outer membrane of chloroplasts and mitochondria. The large conductance
channels of the outer envelope membrane of chloroplasts of Nitella are cation selective ( P
The VDAC found in the outer
membrane of mitochondria also belong to the porin family
(20, 21, 22) . Like the 525-pS channel, it is
weakly anion selective and voltage dependent. However, our study
provides evidence that there are at least five differences between the
VDAC and the 525-pS channel. First, the single-channel conductance of
2.1 nS calculated in 1 M KCl is lower than the values reported
for mitochondrial VDAC (3.7-4.5 nS). Second, the lifetime of the
fully open state of the 525-pS channel is much shorter than that of the
VDAC, which is estimated to be at least several seconds
(20, 21, 22) . Third, the 525-pS channel exists
in three stable conformations: a fully open state, a fully closed
state, and a substate. Thus, the 525-pS channel closes to a
nonconducting state, whereas the mitochondrial VDAC almost never closes
completely and remains either in the fully open state or in the
substate. Fourth, the polyanion dextran sulfate (6.3 and 31
µM) has no effect on the conductance and the gating of the
525-pS channel. In contrast, electrostatic interactions between dextran
sulfate and the positive gating charges of the mitochondrial VDAC
decrease its open probability
(23) . Finally, the voltage gating
of the 525-pS channel depends on the polarity (Fig. 5); the open
probability of the fully open state is higher at positive voltages than
at negative voltages, and the substate appears only at positive
potentials whereas the open probability of the mitochondrial VDAC is
identical whatever the sign of the electrical potential differences and
follows a ``bell-shaped'' function.
To investigate the
gating process of the 525-pS channel, we performed chemical
modifications of the channel with succinic anhydride. Succinylation of
proteins is known to convert the positive groups of some amino acids
into negative groups
(18) . The walls within the pore are
probably lined with such positive residues. When we modified the net
charge of the pore by converting some of the positive amino acids into
negative amino acids, the voltage gating was changed and the open
probability of the channel was dramatically increased. However, it did
not prevent the channel closure at high positive potentials. These
results suggest either that the gating of the chloroplast porin is
directly mediated by the positive charges distributed inside the pore
or that succinylation of some positive residues just stabilizes the
channel conformation in its fully open state.
The conversion of
charges also alters the ion selectivity of the channel. When the
positive residues lining the pore are converted into negative residues
then the local electrostatic force is modified, and the selectivity of
the channel is reversed. These results agree with those observed in the
bacterial porin PhoE and in the mitochondrial VDAC
(19, 24, 25) and suggest that the positive
residues form the filter lining of the pore.
The occurrence of a
porin channel in the inner envelope membrane of chloroplasts is
surprising since such a large channel would dissipate the
electrochemical gradient. It has been shown that the inward side of
vesicles will, after fusion, face the solution bathing the trans
compartment
(26) . Since the inner envelope vesicles are
predominantly inside-out
(27) , the channels will have their
stroma surface facing the cis side of the bilayer. Therefore, under
physiological conditions, the channel should be mainly closed because
the electrical potential of inner envelope membrane was measured to be
about
We are greatly indebted to Prof. J. M. Ruysschaert for
critically reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/ P
= 1.6 ±
0.2). The gating mechanism of the pore is voltage dependent. The
channel shifts from a fully open state to a substate at positive
electrical potentials and remains closed at negative electrical
potentials. Succinylation of the protein increases the open probability
of the fully open state and reverses the channel selectivity. Analysis
of the single-channel conductance as a function of the salt
concentration and of the open probability at various voltages suggests
that this channel is a new membrane porin not previously identified.
(
)
(in 100 mM KCl) was assumed to be
responsible for the high permeability of this membrane
(2) .
Three other channels have been identified in the outer membrane of
giant chloroplasts of Nitella (3) : two are cation
selective channels with a conductance of 520 and 1 nS (in 100
mM KCl) and one 160 pS channel is anion selective. The voltage
dependence and the conductance of the three large channels are similar
to that of the mitochondrial porin, also called the voltage-dependent
anion channel of the mitochondrial outer membrane (VDAC)
(4) .
-ATPase
(5) and several
specific carrier proteins. Two of them have been purified: the
phosphate translocator
(6) and the glycolate transporter
(7) . Little is known about ion channels in the inner envelope
membrane. The pH gradient across the inner envelope membrane is thought
to be regulated by an active extrusion of H
balanced
by a passive counterflux through H
and K
channels
(8, 9) . An inner envelope K
channel has been recently reconstituted into a planar lipid
bilayer
(10) . This channel could be involved in the pH
regulation of the stroma. An anionic flux through the inner envelope
membrane was also detected from swelling studies
(11) and
tracer flux experiments
(12) done with intact chloroplastic
suspensions. The chloride transporter could be the phosphate
translocator, which can also work as an anion channel
(13) .
Purification of the Inner Envelope
Membrane
Inner envelope membranes of chloroplast were
purified from Spinacia oleracia by step-gradient
centrifugation as described by Keegstra and Yousif
(14) .
Spinach leaves were purchased from a local market. About 500 g were
homogenized with a Waring blender in 1 liter of grinding medium (GM)
(0.33 M sorbitol, 1 mM EDTA, 1 mM
MgCl, 50 mM Tricine-KOH, pH 7.8, and 0.1% bovine
serum albumin). All operations were carried out at 4 °C in a cold
room. The mixture was filtered through eight layers of cheese cloth and
one layer of cotton wool, and then 4
250 ml were centrifuged at
2,500
g for 2 min (Sorvall GSA rotor). The pellet was
collected, resuspended in 2
10 ml of GM, and layered over 2
15 ml of 40% (v/v) Percoll (Pharmacia Biotech Inc.) in GM. The
two tubes were centrifuged at 2,000
g for 2.5 min and
then brought to rest without braking (Heraeus, swinging buckets). The
broken chloroplasts and contaminant organelles that formed a band above
the pellet were discarded. Purified intact chloroplasts were recovered
at the bottom of the tube and washed two times in GM without bovine
serum albumin at 2,700
g for 7 min (Sigma centrifuge).
The integrity of the chloroplasts was higher than 95%, as determined
with a Clark-type oxygen electrode
(15) . Organelles (25 mg of
chlorophyll) were resuspended in hypertonic medium (0.6 M
sucrose, 2 mM EDTA, and 10 mM Tricine-KOH (pH 7.8))
and were incubated on ice for 15-20 min at 1 mg/ml chlorophyll.
Then, inner and outer envelopes were detached by 30 strokes with a
Dounce homogeneizer. The mixture was diluted with two volumes of 2
mM EDTA and 10 mM Tricine-KOH (pH 7.8) to give 0.2
M sucrose (final). Thylakoids and whole intact chloroplasts
were removed by a centrifugation at 6,000
g for 10 min
(Sorvall SS34 rotor). The yellow supernatant was collected and pelleted
at 40,000
g for 30 min (Sorvall SS34 rotor) to obtain
the crude envelope. The pellet was suspended in 2
20 ml of 0.2
M sucrose, 2 mM EDTA, and 10 mM Tricine-KOH
(pH 7.8) and layered on the top of a discontinuous sucrose gradient
(0.46 M (1.06 g/ml), 0.8 M (1.10 g/ml), and 1
M sucrose (1.13 g/ml)) in 2 mM EDTA, 10 mM
Tricine-KOH (pH 7.8) and centrifuged at 120,000
g overnight (Beckman SW27 rotor). Outer and inner envelope fractions
formed two bands, which were recovered separately at the second and
third interfaces of the sucrose gradient, respectively. The outer and
inner envelopes were diluted four times with 2 mM EDTA, 10
mM Tricine-KOH (pH 7.8) and were collected after a
centrifugation of 125,000
g for 90 min (Beckmann SW27
rotor). The two pellets were resuspended with a Dounce homogeneizer in
GM. The absence of cytochrome c oxidase activity and
chlorophyll indicated that there was no detectable contamination by
mitochondrial membranes and thylakoids. Small aliquots were stored at
70 °C. Intact chloroplasts equivalent to 1 mg of chlorophyll
yielded about 10 µg of inner envelope proteins and about 5 µg
of outer envelope proteins. The experiments reported in this paper were
carried out using five batches of membrane purified during one year.
Electrical Measurements in Planar Lipid
Bilayers
Planar lipid membranes were formed using the
Mueller-Rudin technique
(16) from a binary mixture of POPE/POPS
(7:3 (w/w), Avanti Polar Lipids, AL) dissolved in n-decane.
Experiments were performed at room temperature. All solutions of triple
distilled water were millipore filtered. The two chambers were
connected to the voltage generator through Ag/AgCl electrodes and a
bridge of 3 M KCl, 2% agar. The current was measured using a
Biologic RK-300 patch-clamp amplifier (Claix, France) and stored in a
digital form on a videotape. The data were filtered with a low-pass
5-pole Tchebicheff filter at a cut-off frequency of 300 Hz. Acquired
data were subsequently replayed, and the relevent events were sampled
at 1 kHz and stored in a 386-Olivetti computer. Electrical potentials
were defined as cis with respect to trans, which was held at ground.
) was
calculated according to the equation
(17) P
=
(1/ N
)
P
,
where
is the summation index from 0 to
N
, P
is the fraction of time
during which a channel opens, and N
is the total
number of channels in the planar lipid bilayer. In the absence of a
substate, the channel behaves like a two-state channel. In this case,
the open probability was estimated from records containing one or two
channels. In contrast, in the presence of the substate, the open
probability of the fully open state and the open probability of the
substate were estimated from the fraction of time spent in the fully
open state or in the substate. In this case, experiments containing
only one channel were used for the analysis.
, 10 mM
Hepes-KOH, pH 7.2; trans side: 10 mM Hepes-KOH, pH 7.2) and an
electrical potential of +60 mV across the bilayer. After fusion,
an aliquot of 3 M KCl was added in the trans side (150
mM KCl, final concentration).
Chemical Modification
Succinic anhydride
(Sigma) was added from a stock solution of dimethyl sulfoxide to both
cis and trans compartments to a final concentration of 3.8 mM.
The compartments were stirred during 30 s, and measurements were
performed after waiting for 5 min of incubation for a complete
hydrolysis of succinic anhydride (the anhydride half-life is about 2
min). No electrical potential was applied across the bilayer during the
incubation of the ion channel with succinic anhydride. Control
experiments showed that the conductances of pure lipid bilayers were
not modified by MeSO alone or 3.8 mM succinic
anhydride, but addition of higher concentration of anhydride
destabilized the planar bilayer. We used a 150 mM KCl solution
buffered with 50 mM MOPS-KOH, pH 7.2, to prevent a pH decrease
during hydrolysis of succinic anhydride, which could affect the
anhydride reaction with amino groups
(18) . In experiments
carried out in a 1 M, 0.1 M KCl gradient, no succinic
anhydride was added in the low salt compartment (the trans side) to
minimize increase of salt buffer due to the presence of MOPS.
Conductance and Selectivity of the
Channel
Isolated inner envelope membrane of chloroplasts
are incorporated into a planar lipid bilayer separating two aqueous
solutions. After addition of membrane vesicles to the cis side of the
planar lipid bilayer and stirring for a maximum of 30 min, we sometimes
observed a discrete jump of currents in the electrical record
reflecting fusion between a vesicle containing one channel and the
planar bilayer. Channels were successfully reconstituted in about 20%
out of the trials. One channel of large conductance was reliably
observed in planar bilayers enriched with the inner envelope. This
channel was detected in 63 out of 84 successful reconstitutions of the
inner envelope fractions. It is quite specific of the inner envelope
and was never observed when the reconstitution was carried out with the
thylakoids ( n > 50) or the outer envelope fraction ( n > 20). The channel remained mainly closed at negative
potentials (Fig. 1). At positive potentials, the channel opened
more frequently. Besides the transition between a fully open and a
fully closed state, the channel shifted from its open state to a
substate at +50 and +60 mV. Fig. 2 A shows the
I-V relationship of the fully open state in symmetrical 150 mM
KCl. A slope conductance of 525 ± 12 pS ( n = 26)
is calculated between ±40 mV from the linear regression of the
ohmic part of the curve. The current characterizing the substate is
about half that the maximum opening (Fig. 2 B).
Figure 1:
Typical current fluctuations of a
single channel from the inner envelope of chloroplasts. The inner
envelope membrane was incorporated into a planar lipid bilayer of
POPE/POPS (7:3, w/w). The electrical measurements were carried out in
symmetrical solution (150 mM KCl, 10 mM Hepes-KOH (pH
7.2)). The electrical potential difference (mV) is indicated at the
right of each current trace. The closed state is indicated by
c. Positive currents flowing from the cis to the trans side
are plotted downward. The arrows indicate the substate of the
channel.
Figure 2:
I-V
relationships of the channel. A, the I-V relationship was
measured in symmetrical conditions (150 mM KCl, 10 mM
Hepes-KOH (pH 7.2)). The data from 26 independent experiments were
fitted by a third-order polynomial regression ( r= 1). The slope conductance of the ohmic part of the curve
was calculated from a linear regression between
40 and +40
mV for each of the 26 independent experiments. The r
coefficients were higher than 0.99. The conductance of the
channel is 525 ± 12 pS. B, the substates were observed
only at positive potential differences. The I-V relationship of the
substate (
) was fitted by a third-order polynomial regression
( r
= 0.985). The I-V curve of the fully
open state was redrawn from A (
).
The
use of different lipid composition such as POPE, POPE/palmitoyloleoyl
phosphatidylcholine (1:1, w/w), diphytanoyl phosphatidylcholine, or
azolectin has no effect on both frequency of reconstitution and
conductance of the channel. The electrical properties of the 525-pS
channel measured in the presence of divalent cation in the cis
compartment (5 mM Caor 5 mM
Mg
) were not affected by the presence or the absence
of 10 mM EGTA.
3.9 mV
( n = 2), and +8.5 mV ( n = 1),
respectively (Fig. 3). The selectivity of Cl
over K
in these gradients is 1.6 ± 0.2
( n = 7).
Figure 3:
The selectivity of the channel. The
selectivity was calculated from the reverse potential measured in three
salt gradients buffered with 10 mM Hepes-KOH (pH 7.2). The
ionic concentrations in the cis/trans compartments were 450/150
mM KCl (), 150/400 mM KCl (
), and 1000/150
mM KCl (
). Data were fitted by a third-order
polynomial regression (
, r
= 0.998;
, r
= 0.999;
,
r
= 0.993).
Effects of the Salt Concentration on the Channel
Conductance
Single-channel conductance is measured in
symmetrical KCl concentrations ranging from 50 mM to 1
M. The I-V curves measured in salt concentrations of 1
M, 750 mM, and 500 mM KCl can be fitted by a
linear relationship (Fig. 4 A). The linear regressions
give single-channel conductances of 2.1, 1.7, and 1.2 nS, respectively.
At lower salt concentrations (50 and 100 mM KCl), the slope
conductances calculated between 40 and +40 mV are 168 and
349 pS, respectively (Fig. 4 B).
Figure 4:
Effect of salt concentration on the
single-channel conductance. A, the I-V curve was measured in a
symmetrical 150 mM KCl, and then KCl concentration was
increased to the required concentration by the addition of aliquots of
concentrated KCl to each compartment. Each curve represents
data points from an independent experiment. The slope conductance was
calculated by linear regression. The rvalues were
higher than 0.995. Single-channel conductances are 2.1 nS in 1
M KCl (
), 1.7 nS in 750 mM KCl (
), and
1.2 nS in 500 mM KCl (
).
represents the current
measured in symmetrical 150 mM KCl and redrawn from Fig.
2 A. B, the channel was reconstituted in a salt
gradient of 50 mM KCl, 10 mM Hepes-KOH (pH 7.2) in
the cis compartment and 10 mM Hepes-KOH (pH 7.2) in the trans
compartment. Then, the KCl concentration was increased to make a
symmetrical final concentration of either 50 or 100 mM KCl.
Each curve represents data points from an independent
experiment. The slope conductances were calculated by a linear
regression from
40 to +40 mV ( r
>
0.990). The conductances were 349 pS in 100 mM KCl (
and 168 pS in 50 mM KCl (
).
represents the current
measured in symmetrical 150 mM KCl from Fig. 2 A.
C, the slope conductance is plotted as a function of the
conductivity of the experimental solution. Data were fitted with a
linear regression ( r
=
0.998).
Large channels called
porin form water-filled pores across the membrane through which small
solutes can diffuse
(19, 20, 21, 22) .
Their single-channel conductance increases linearly with the solute
concentration. This characteristic is also shared by the 525-pS channel
whose single-channel conductance is found to be a linear function of
the conductivity of the aqueous solution (Fig. 4 C).
Voltage-dependent Gating of the
Channel
The study of the voltage dependence is performed in
symmetrical 150 mM KCl, 10 mM Hepes-KOH (pH 7.2).
Fig. 5A illustrates the open probability of the fully
open state at various voltages compiled from 13 bilayers. The channel
exhibits an open probability of about 0.2 at voltages ranging from
+15 to +60 mV. The slight decrease of the open probability
observed after +60 mV is not statistically significant. At
negative potentials, the open probability decreases to zero. The open
probability of the substate is shown in Fig. 5 B. The
substate becomes a long-lived state after +40 mV. The open
probabilities of the substate calculated at +40 and +60 mV
are statistically different.
Figure 5:
Voltage dependence of the channel. The
experiments were performed in symmetrical 150 mM KCl, 10
mM Hepes-KOH (pH 7.2). Single-channel open probabilities were
estimated using digitized data of either 30- or 60-s durations.
A, the open probability of being in the fully open state
measured at various voltages was compiled from 13 bilayers. B,
the open probability of being in the substate measured at various
voltages was obtained from 10 bilayers.
Chemical Modification of the Channel
To
investigate the gating process of the channel, we used succinic
anhydride to convert a positive amino group into a negative carboxyl
group
(18) . The bilayer lipid membrane is formed in a
symmetrical 150 mM KCl solution, and succinic anhydride (3.8
mM, final concentration) is added on each side of the
membrane. 5 min after addition of succinic anhydride, the channel is in
its open state and closes only on rare occasions when the membrane is
polarized at 30 mV (Fig. 6 A). When a positive
membrane potential difference of +50 mV is applied, the modified
channel remains in the open state for few seconds and afterward
completely closes (Fig. 6 A). Fig. 6 B summarizes the open probabilities of the fully open state measured
before and after succinylation of the channel. At electrical potential
differences ranging from
70 to +40 mV, the open probability
reaches the maximum ( P
= 1). But at
electrical potentials higher than +40 mV, the open probability
decreases because the channel shifts either to the substate or to the
fully closed state. The succinylation does not seem to change the
structure of the pore since the conductance is not affected by the
chemical treatment. To measure the effect of succinic anhydride on the
ion selectivity of the channel, we have reconstituted ion channels in
planar bilayers in the presence of a salt gradient of 1000/100
mM KCl (cis/trans). The I-V relationship is recorded before
and after addition of succinic anhydride (3.8 mM) to the cis
side. The reversal potential shifts from +8.4 mV ( n = 2) to
8.9 mV ( n = 2) when the
channel is succinylated (Fig. 7), which indicates that
succinylation of the channel changes its selectivity. The anionic
channel becomes cationic with a selectivity for K
over
Cl
of 0.67 instead of 1.6 calculated for the
unmodified channel.
Figure 6:
Effect of succinic anhydride on the open
probability of the fully open state. Succinic anhydride (3.8
mM) was added to the cis and trans compartments of a bilayer
formed in symmetrical 150 mM KCl and 50 mM MOPS-KOH,
pH 7.2. After 5 min of treatment, Pwas determined
at various membrane potentials using recordings of either 30- or 60-s
durations. Control experiments were performed before addition of the
chemical. A, the current traces are typical recordings of the
channel activity at
30 and +50 mV. The fully closed state
is indicated by c. B, the open probability of the
fully open state of the channel treated with succinic anhydride
(
) is compared with that of the unmodified channel (
).
Data are collected from two independent bilayers (
) or are the
mean calculated from three independent bilayers
(
).
Figure 7:
Effect of succinic anhydride on the ion
selectivity. The selectivity of the channel was calculated from the
reverse potentials measured in a salt gradient (cis/trans) (1
M KCl, 50 mM MOPS-KOH, pH 7.2, 100 mM KCl,
10 mM Hepes-KOH, pH 7.2). The I-V relationships before
() and after (
) addition of 3.8 mM succinic
anhydride were measured for two independent bilayers. Data were fitted
by a linear regression ( r
= 0.994 and
0.991, respectively).
Dextran sulfate is a large impermeant polyvalent
anion (8 kDa) known to decrease the open probability of the
mitochondrial VDAC at a concentration of 6 µM(23) . To test if dextran sulfate is a potent inhibitor of the
525-pS channel, it was added on each side of the bilayer (6.3 and 31
µM, final concentrations). Neither the voltage dependence
nor the conductance of the 525-pS channel were modified whatever the
concentration used (data not shown). Each experiment was repeated four
times.
/ P
= 4.6 and 3.5)
(3) , and that of spinach
chloroplast has a conductance of 7 nS in 1 M KCl, which is one
of the largest conductances of all known porins
(2) . The
probability that the 525-pS channel observed in the inner envelope
fraction could be a contamination from the thylakoid membrane or from
the outer envelope is very weak. Indeed, the electrical signal
characterizing the 525-pS channel was never observed when the outer
envelope membrane or the thylakoid membrane of the chloroplast was used
instead of the inner envelope fraction.
100 mV
(28) . It cannot be ruled out that the
525-pS channel might remain closed by soluble elements present in the
chloroplast stroma or inter-envelope space. Large conductance channels
involved either in pressure transduction or in import of proteins have
been already observed in the inner membrane of mitochondria and
bacteria
(29, 30, 31, 32, 33, 34) .
The reconstitutions of contact sites from brain mitochondria and
Escherichia coli have shown that these membrane fractions
contain several ion channels with a conductance ranging from 475 pS to
1 nS in 150 mM KCl
(35) and from 300 pS to 1.5 nS in
100 mM KCl
(30) . The exact mechanism of protein
transport across the envelope membranes of chloroplasts is still
unknown. It has been suggested that protein translocation proceeds via
two distinct protein-conducting channels in the outer and in the inner
envelope membranes
(36) . Therefore, an alternative explanation
could be that the 525-pS channel is a component of the protein import
machinery present in the inner envelope of chloroplasts.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.