(Received for publication, February 14, 1995; and in revised form, April 27, 1995)
From the
Spinach leaf peroxisomes were purified by Percoll density
gradient centrifugation. After several freeze-thaw cycles, the
peroxisomal membranes were separated from the matrix enzymes by sucrose
density gradient centrifugation. The purity of the peroxisomal
membranes was checked by measuring the activities of marker enzymes and
by using antibodies. Lipid bilayer membrane experiments with the
purified peroxisomal membranes, solubilized with a detergent,
demonstrated that the membranes contain a channel-forming component,
which may represent the major permeability pathway of these membranes.
Control experiments with membranes of other cell organelles showed that
the peroxisomal channel was not caused by the contamination of the
peroxisomes with mitochondria or chloroplasts.
The peroxisomal
channel had a comparatively small single channel conductance of 350 pS
in 1 M KCl as compared with channels from other cell
organelles. The channel is slightly anion selective, which is in
accordance with its physiological function. The single channel
conductance was found to be only moderately dependent on the salt
concentration in the aqueous phase. This may be explained by the
presence of positive point net charges in or near the channel or by the
presence of a saturable binding site inside the channel. The possible
role of the channel in peroxisomal metabolism is discussed.
Leaf peroxisomes belong to the microbodies, a group of small
multipurpose cell organelles which are found in all, except some very
primitive, eukaryotic cells(1) . They are surrounded by a
single membrane. Peroxisomes usually, although not always, contain
H
Leaf peroxisomes play a vital role
in photosynthesis. They are involved in the recycling of glycolate
formed as an unavoidable by-product of CO
The results are conflicting as to
whether other types of peroxisomes contain a pore-forming protein or
specific translocators. Liver peroxisomes were found to be permeable to
sucrose and nucleotides(10) . Upon reconstitution experiments a
channel forming activity was attributed to a 22-kDa membrane
popypeptide(11) , but the subsequent analysis of the amino acid
sequence did not show any similarity to known porin
structures(12) . Other experiments incorporating membrane
preparations of liver peroxisomes into liposomes, using the patch clamp
technique(13) , and into planar lipid bilayers (14) indicated that large cation-selective voltage-gated pores
with an estimated diameter of 1.5-3.0 nm may be responsible for
the high permeability of liver peroxisomes. In the yeast Hansenula
polymorpha, a 31-kDa peroxisomal integral membrane protein, with a
structure similar to the 31-kDa mitochondrial porin of Saccharomyces cerevisiae, was claimed to be responsible for
the in vitro permeability of the peroxisomes(15) .
Some results are not consistent with the presence of a free
diffusion channel in peroxisomes. Yeast peroxisomal membranes contain a
proton-translocating ATPase(16) , and a proton gradient across
the peroxisomal membrane has been observed(17) . An ATPase
activity was also found in the membranes of liver
peroxisomes(18) . An integral membrane protein showing a high
structural homology with mitochondrial anion translocators (19) has been identified in the peroxisomal membrane of the
yeast Candida boidinii. This suggests that these peroxisomes
contain a specific metabolite translocator. The boundary function of
the peroxisomal membrane is thus still a matter of debate.
We set
out to investigate whether leaf peroxisomal membranes contain channel
forming activity. This report shows that leaf peroxisomal membranes
contain channels allowing the passage of the metabolites of the
photorespiratory metabolism and which are distinctly different from the
porin channels of leaf mitochondria and chloroplasts.
Figure 1:
Subfractionation of the peroxisomal
suspension on a sucrose gradient. The gradient fractions were analyzed
for protein and sucrose concentration (A) and enzyme activity
of the peroxisomal matrix enzymes hydroxypyruvate reductase (HPR) and catalase (CAT) (B), the
peroxisomal membrane enzyme ACS (C), and cytochrome c reductase (CCR) and ferricyanide reductase (FR) (D). Results are expressed as percentage of total gradient
activity in each fraction. Fractions 1 and 12 represent the top and the
bottom fractions, respectively. Recoveries of protein and marker
enzymes (recovery, total gradient protein, or activity) were checked
(protein: 101%, 11.7 mg; hydroxypyruvate reductase: 80%, 114
µmol
Figure 2:
Western blot analysis as control for
membrane contamination of the peroxisomal membrane fractions and
SDS-PAGE. Fractions of the sucrose gradient were subjected to SDS-PAGE (A) and immunoblotted with a polyclonal antibody against the
24-kDa protein of the outer envelope membrane of spinach chloroplasts (B) or with a polyclonal antibody against the 30-kDa porin of
pea root plastids, showing strong cross-reactivity with the 30-kDa
porin of the outer membrane of spinach mitochondria (C) as
described under ``Experimental Procedures.'' For Western blot
analysis in each lane, 20 µg of protein and for silver stain 3
µg of protein were separated by SDS-PAGE. Fractions 8+9 and 10+11 were pooled in a protein ratio of 1:1
because of their minor protein content. As a control 5 and 20 µg of
protein of spinach chloroplast envelope membranes and mitochondrial
membranes were blotted. P, peroxisomal
suspension.
Figure 6:
Comparison of porin activity and content
of peroxisomal membrane in the fractions of the sucrose gradient. The
concentration of peroxisomal membrane was measured on the basis of the
specific ACS activity. The channel-forming activity was determined as
explained in the text by using membranes from diphytanoyl
phosphatidylcholine/n-decane. The voltage applied was 10 mV; T =
25 °C.
Mitochondria were purified
according to the method of Neuburger et al.(23) . The
mitochondria were disrupted osmotically by incubation for 30 min in
distilled water on ice and sedimented afterward (160,000
In the
experiment shown in Fig. 1, the activities of marker enzymes for
the various subcellular components have been measured in the different
fractions of the sucrose density gradient. Most of the protein (about
80%) and the marker enzyme for the peroxisomal matrix catalase and
hydroxypyruvate reductase are found in the upper part of the gradient.
These fractions represent the peroxisomal matrix proteins. The ACS
activity forms a distinct peak at a density of 1.21-1.23 g/ml.
Control experiments by measuring specific marker enzymes and Western
blot analysis of all fractions of the sucrose gradient excluded that
this ACS peak resulted from nonperoxisomal sources. 1) One of the major
constituents of the outer envelope membrane of spinach chloroplasts is
a 24-kDa protein which function is unknown until now(46) .
Using an antibody against this protein, we could localize the
contaminating outer envelope membrane in the first fraction of the
sucrose gradient (Fig. 2B) at its low equilibrium
density concurring with earlier results(39, 47) . The
24-kDa protein was not detectable in the fractions containing ACS
activity indicating that the outer envelope membrane is absent in these
fractions. 2) NADH-cytochrome c reductase, a marker enzyme for
ER membranes(25) , was found at low activities on the top of
the gradient at a density of 1.15 g/ml as shown earlier(25) .
3) The outer membrane of mitochondria was detected with the marker
enzyme NADH-ferricyanide reductase (22) and in addition by
using polyclonal antibodies against the 30-kDa porin of non-green pea
root plastids(9) . As the porins of plant non-green plastids
and mitochondria are relatively homologous proteins(9) , these
antibodies show cross-reaction with the mitochondrial 30-kDa porin of
spinach (Fig. 2C) and are thus a useful marker for the
detection of the outer membrane of spinach mitochondria. Using both
methods we were able to show that the content of the outer
mitochondrial membrane in that part of the gradient where the maximal
ACS activity is localized is rather low. The cross-reaction with a
polypeptide of 66 kDa in fractions 10-12 could be an nonspecific
artifact. The activity of NADH-ferricyanide reductase in the upper part
of the gradient (fractions 1-4) may be due to peroxisomal
activity as potato tuber peroxisomes were shown to possess this
enzyme(48) . Thylakoid membranes were enriched at
1.17-1.18 g/ml (data not shown) as reported earlier(49) .
As the ACS peak contained neither outer chloroplast envelope
membranes, outer mitochondrial membranes, nor ER membranes to any
detectable extent, it can be concluded that the ACS peak represents the
peroxisomal membrane and that this membrane is not contaminated with
outer membranes of chloroplasts or mitochondria to any appreciable
amount. As the outer mitochondrial and outer chloroplast envelope
membranes both contain porins, the absence of these membranes in the
peroxisomal membrane fraction is essential. In gradients with
incompletely disrupted peroxisomes, the peroxisomal membrane could be
identified from the adhering activities of the peroxisomal enzymes
catalase, hydroxypyruvate reductase, and malate dehydrogenase, forming
a second smaller peak at 1.215 g/ml. Glyoxysomal membranes have been
reported to equilibrate at this density(50) . SDS-PAGE was
performed with the fractions 1-12 of the same gradient (Fig. 2A). The dominant proteins of the peroxisomal
membrane had a subunit molecular mass of about 50, 48, 45, 43, 39, 32,
and 13 kDa.
Figure 3:
Single channel recording of a diphytanoyl
phosphatidylcholine/n-decane membrane in the presence of
detergent-solubilized spinach leaf peroxisomes. 20 min after the
formation of the membrane 1 µg/ml detergent-solubilized spinach
leaf peroxisomes was added to the aqueous phase on one side of the
membrane. The aqueous phase contained 1 M KCl (pH 6). The
applied membrane potential was 10 mV; T = 25
°C.
Figure 4:
Histogram of the probability of the
occurrence of certain conductivity units observed with membranes formed
of diphytanoyl phosphatidylcholine/n-decane in the presence of
1 µg/ml detergent-solubilized spinach leaf peroxisomes. The aqueous
phase contained 1 M KCl. The applied membrane potential was 10
mV; T = 25 °C. The average single channel
conductance was 350 pS for 344 single channel events. The data were
collected from 10 different membranes. P(G), probability of
the single channel conductance G.
Figure 5:
Channel formation by peroxisomal membranes
taken from a sucrose density gradient. Stepwise increase of the
membrane current given after the addition of Genapol-solubilized
peroxisomal membranes (A) to a black lipid bilayer membrane
given as a function of time and comparison with chloroplast envelope (B) and mitochondrial porin activity (C). In A the peroxisomal membrane fraction of the sucrose gradient was
added. Sometimes the incorporation of larger pores, similar to
described chloroplast envelope porins (B) and mitochondrial
porins (C), was observed. The aqueous phase contained about 1
µg/ml protein and 1 M KCl. The membrane was formed from
diphytanoyl phosphatidylcholin/n-decane. The voltage applied
was 10 mV; T = 25 °C.
The
channel formed by peroxisomal porin was permeable for a variety of
ions. Table 2summarizes the single channel conductance for
different salt solutions. The nature of the anions had a substantial
influence on the single channel conductance, whereas the influence of
the cations was rather small. Thus, the single channel conductance in 1 M KCl was approximately the same as in 1 M LiCl, but
it was considerably smaller in 1 M potassium acetate
(K
We thank Dr. S. Borchert for useful discussions and M.
Raabe for her excellent help in preparation of peroxisomes. The
antibodies were kindly provided by Prof. Dr. Flügge
(Universität Köln).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O
producing enzymes and catalase to eliminate
the H
O
(2) . They all contain the
enzymes for
-oxidation of fatty acids. Present evidence suggests
that peroxisomes are not formed de novo but grow and divide
like plastids and mitochondria, although they have no genome of their
own. There are indications that all peroxisomes, e.g. from
fungi, higher plants, and animals, have a common origin, possibly an
endosymbiotic event(2) .
fixation due to
oxygen reacting instead of CO
with ribulose bisphosphate.
In a leaf the ratio of oxigenation/carboxylation during photosynthesis
is between 0.2 and 0.5, resulting in very high metabolic fluxes through
the leaf peroxisomes. The peroxisomal reaction chains are strictly
compartmentalized. When leaf peroxisomes were subjected to an
``osmotic shock'' the peroxisomal membrane was damaged
extensively, but surprisingly the peroxisomal matrix did not
disintegrate and its metabolic function, including the high
compartmentation of metabolism, was unaltered(3, 4) .
The intermediates of the reaction chains did not leak out(5) .
From these findings we concluded that the compartmentation of
metabolism in leaf peroxisomes is not due to a boundary function of the
peroxisomal membrane but is the result of the properties of the
peroxisomal matrix which allow metabolite channelling(3) . Our
results suggest that specific translocators are not essential for the
functional compartmentation of metabolism. Nonspecific pores seem
sufficient for the metabolite transfer into and out of the leaf
peroxisomes. General diffusion channels, called porins, exist in the
outer membrane of Gram-negative eubacteria (for review, see (6) ), mitochondria(7) , and
plastids(8, 9) . Bacterial porins occur as trimers of
three identical subunits. Each subunit contains one diffusion channel
for small molecules. They are formed entirely of amphipathic
-sheets arranged in a barrel-like
structure(6, 7) .
Plant Material
Peroxisomes, mitochondria, and
chloroplasts were isolated from leaves of spinach (Spinacia
oleracea L., U.S Hybrid 424; Ferry-Morse Seed Company, Mountain
View, CA). The plants were grown and harvested as
described(4) .
Isolation of Leaf Peroxisomes
Peroxisomes were
isolated by modifying to the method of Yu and Huang(20) . The
scale was increased by a factor of five up to 350 g of leaves, and the
homogenization procedure was intensified. The final peroxisomal pellet
was resuspended and stored at -20 °C. The yield of
peroxisomes was about 6 mg of protein with a specific activity of
hydroxypyruvate reductase of about 19
µmol(min
mg)
(see Table 1). The
intactness measured as latency of hydroxypyruvate reductase (3) was about 95%.
Isolation of Leaf Peroxisomal Membranes
The highly
aggregated structure of the matrix enzymes was destroyed by subjecting
the organelles (12 mg of protein) to five freeze-thaw cycles (freezing
in liquid nitrogen, thawing at room temperature) and intensive
homogenization in a Potter homogenizer. The suspension was adjusted to
30% (w/w) sucrose, loaded on a linear sucrose gradient (35-60%
(w/w) sucrose in 10 mM HEPES, pH 7.5, 0.8 mM MgCl), and centrifuged in a swing-out rotor (240,000
g, 15 h, Sorvall AH 650). The gradient was
fractionated from the top (12 fractions of 0.4 ml). The fractions were
diluted to a sucrose content of 30% (w/w) and stored at -80
°C. All the data of enzyme activities (Fig. 1),
SDS-PAGE
(
)and Western blot analysis (Fig. 2), and porin activity (Fig. 6) were obtained with
the same gradient and confirmed by additional experiments.
min
; catalase: 91%, 11.3
mmol
min
; ACS: 84%, 128
nmol
min
; NADH-ferricyanide reductase: 71%,
3.64 µmol
min
; NADH-cytochrome c reductase: 79%, 44.2
nmol
min
.
Isolation of Chloroplast Envelopes and of Mitochondrial
Membranes
Chloroplasts were isolated according to Heldt and
Sauer (21) and chloroplast envelope membranes according to
Douce et al.(22) .
g, 45 min, Kontron TFT 65.13).
Measurement of Enzyme Activities
If not stated
otherwise, measurements of marker enzyme activities were carried out at
25 °C in a final volume of 1 ml. Hydroxypyruvate reductase and
catalase were measured as described previously(3) . Acyl-CoA
synthetase (ACS) was measured as described by Fischer et
al.(9) . The reaction was stopped after incubation for 10
min at 30 °C. The measurement of NADH- ferricyanide reductase (700
µl)(24) , NADH-cytochrome c reductase(25) , cytochrome c oxidase (700 µl) (24) , and NADP-glycerinaldehyde-3-phosphate dehydrogenase (26) was performed as described. Protein was determined
according to Peterson (27) .
SDS-PAGE and Western Blot Analysis
Precipitation
of protein fractions was carried out using
chloroform-methanol(28) . SDS-PAGE was performed on 12.5% gels
according to Laemmli(29) . Polypeptide bands were made visible
by silver staining(30) . In Western blot analysis the bound
antibodies were made visible with a peroxidase-coupled second antibody
using an ECL-kit (Amersham-Buchler, Braunschweig, Germany).
Lipid Bilayer Experiments
The methods used for the
bilayer experiments have been described in
detail(31, 32) . Peroxisomal membranes were
solubilized in 0.5% Genapol X-80 (Fluka, Neu-Ulm) and added to the
aqueous phases at one or both sides of the black membranes.
Preparation of Peroxisomes
From measurement of
the activities of hydroxypyruvate reductase and catalase as marker
enzymes for the peroxisomal matrix, the yield of the peroxisomes
obtained from spinach leaves was evaluated as about 5 and 11% (Table 1). This low yield reflects the difficulties of
peroxisomal isolation and is inherent to all published preparation
procedures(20, 33) . The contamination of the
peroxisomal preparation with chloroplasts, mitochondria, and ER is
quite low, as the recoveries of the corresponding marker enzyme
activities were 0.05, 0.2, and 0.01% as compared to the starting
homogenate (Table 1). From this the contamination of the
peroxisomal fraction by mitochondria, chloroplasts, and ER can be
evaluated as less than 1, 4, and 0.2%, respectively. The purity of the
peroxisomal suspension has been checked earlier by electron
microscopy(3) .
Isolation and Purification of the Peroxisomal
Membrane
The most used method for the isolation of peroxisomal
membranes of rat liver (11, 34) and glyoxysomes (35, 36) is treating the peroxisomes with 100 mM sodium carbonate at pH 11.5(37) . This method was
unsuitable because our measurements indicated a destruction of the
porin activity. After various attempts to solubilize the highly
aggregated matrix enzymes, we found it best to disrupt the peroxisomes
mechanically (see ``Experimental Procedures'') and to
separate the membranes by centrifugation in a sucrose density gradient.
At present, unfortunately, there is no specific marker enzyme for the
peroxisomal membrane of leaves. ACS, which has been shown to be closely
associated with the membranes of leaf peroxisomes (38) and
glyoxysomes (a differentiation form of plant peroxisomes), is present
in the outer membrane of the chloroplast envelope (39, 40, 41) and also in the microsomal
membranes(42, 43, 44) . It is possible that
the outer membranes of plant mitochondria also contain this
enzyme(45) . The results of Table 1show that only a
minor portion of the cellular ACS activity is associated with the
peroxisomes. Despite this, because of the very low contamination of the
peroxisomal suspension by other ACS-containing membranes (Table 1) the ACS could be used as marker for the peroxisomal
membrane. If 50% of the ACS activity were associated with the
chloroplasts, the ACS activity from contaminating chloroplasts in the
peroxisomal preparation would be less than 25% of the measured ACS
activity. The same calculation with ER results in 1%.
Solubilized Peroxisomes Show a Pore Forming
Activity
In further experiments we investigated whether
peroxisomes contain any pore forming activity. The sedimented
peroxisomes (protein concentration about 1 mg/ml) were treated with the
detergent Genapol X-80 (final concentration 0.5%) to solubilize the
peroxisomal membrane. The detergent extract was added to the aqueous
phase, bathing a lipid bilayer, and the membrane current was measured. Fig. 3demonstrates that there is indeed a pore forming activity
in these detergent extracts. Each conductance step of Fig. 3corresponds to the incorporation of one channel-forming
unit into the membrane. The average single channel conductance of these
channels is only 350 pS in 1 M KCl (see the histogram in Fig. 4). The occurrence of a single channel conductance of 600
pS probably indicates the incorporation of a dimer. The conductance of
the peroxisomal channel is rather small as compared with those channels
formed by mitochondrial or chloroplast porins under otherwise identical
conditions (see also below).
Identification of the Channel in the Peroxisomal
Membrane
The channel formed by the detergent extracts from whole
peroxisomes had a completely different single channel conductance as
compared with the porins from other plant cell membranes. To give
further direct evidence that the origin of the novel channel-forming
protein is the peroxisomal membrane, we investigated the channel
forming activity of the various fractions of the sucrose gradient as
follows: small quantities of the fractions were dissolved in Genapol
X-80 and added to the aqueous phase on both sides of an artificial
bilayer (final protein concentration in the aqueous phase 0.6
µg/ml). After a lag time of a few minutes, probably caused by slow
aqueous diffusion of the protein, the conductance of the membrane,
caused by the insertion of channels into the membrane, started to
increase. The time course of the increase was similar to that described
previously for porins of mitochondrial or bacterial
origin(51, 52) . The number of inserted pores with a
single channel conductance of less than 1 nS in a given time (20 min)
was counted. The distribution of these conductance steps in a histogram
(data not shown) was similar to that shown for whole peroxisomes (Fig. 4). The mean values of three measurements were taken as a
semiquantitative measure for the peroxisomal channel forming activity
of the sample. After the insertion of more than 30-50 pores in
one black membrane, the single channel conductance could not be clearly
identified (see Fig. 3and Fig. 5). As the channel
incorporation was so frequent for the fractions containing the
peroxisomal membranes, several bilayers had to be painted during the
measuring period. Control experiments showed that the addition of the
detergent Genapol X-80 alone at a similar concentration to that used
with the protein did not lead to any appreciable increase in the
membrane conductance.
We were able to identify three different types
of channels in the protein samples taken from the sucrose density
gradient. The light fractions contained (besides the 350 pS channel;
see Fig. 5A) a channel with a giant single channel
conductance of 7-9 nS in 1 M KCl. This channel was
similar to that found in reconstitution experiments with the outer
membrane of the chloroplast envelope (see Fig. 5C; 8).
In other fractions (preferentially fractions 2-3), we sometimes
observed an additional channel, which was indistinguishable from
mitochondrial porin from plant and other sources (see Fig. 5B; 7). The most prominent channel, however, in
all fractions was the 350 pS channel. Fig. 6shows the
distribution of this channel within the fraction of the sucrose density
gradient. It is noteworthy that the peroxisomal porin activity measured
as the number of channels (20 min after the protein addition) in a
membrane with a surface area of 1 mm corresponded to the
350 pS channel only. When one of the other channels did happen to
incorporate into the lipid bilayer membrane, the experiment was stopped
and a new experiment was started. This was necessary because of the
much higher single channel conductance of the mitochondrial and
chloroplast porins. Fig. 6shows also the specific activity of
ACS, i.e. the content of peroxisomal membrane in the fractions
of the sucrose density gradient. There is a correlation between the
specific ACS activity and the pore forming activity showing that the
350 pS channel resides within the peroxisomal membrane.
Properties of the Peroxisomal Channel
After the
addition of whole peroxisomes or peroxisomal membranes, which had been
solubilized with Genapol X-80, step increases in membrane conductance
could be resolved (see Fig. 3and Fig. 5). The average
single channel conductance with 1 M KCl was 350 pS (see the
histogram of Fig. 4). Fig. 3and Fig. 5show also
that most of the steps were directed upwards. Only a few downward steps
were observed which means that the peroxisomal channels had a long
lifetime. Even at higher transmembrane potentials of about 50 mV the
closing events did not become more frequent. This demonstrates that the
peroxisomal porin is not voltage-regulated at these potentials.
and Cl
and Li
and acetate have the same aqueous mobility)(53) . This
result suggests that the channel had a certain preference for anions.
We found that a variety of organic anions, such as formiate, glycerate,
and glycolate are permeating this channel. It is noteworthy, that the
conductance of the channel was not a linear function of the bulk
aqueous concentration, which may indicate that positive point net
charges are localized in or near the channel mouth. On the other hand,
it is also possible that the channel contains a binding site for
organic anions, which facilitates the diffusion of these ions in a
similar way as specific bacterial porins do for certain substrates such
as sugars, nucleosides, and phosphate(6) .
Ion Selectivity
The single channel data suggested
that the peroxisomal channel is anion-selective. We performed zero
current membrane potential measurements to study its ion selectivity in
more detail. Membranes were formed in 100 mM KCl, and
detergent-solubilized peroxisomal membrane was added to the aqueous
phase when the membranes were in the black state. After incorporation
of 100-1000 channels into a membrane, salt gradients were established
by addition of small amounts of 3 M KCl solution to one side
of the membrane. Under these conditions, the more diluted side of the
membrane became negative which indicated indeed preferential movement
of chloride over potassium. However, the potential and the permeability
ratio between potassium and chloride (as calculated from the
Goldman-Hodgkin-Katz equation; 54) differed considerably from
experiment to experiment, which probably means that other channels such
as mitochondrial porin or the chloroplast porin interfered with the
measurements and led to irreproducible results. This is because the
single channel conductance of the peroxisomal porin (350 pS) is about
6-12 times smaller than that of mitochondrial porin (2.4 or 4.0
nS) and about 23 times smaller than that of chloroplast porin (8 nS)
under otherwise identical conditions (1 M KCl).
The Peroxisomal Channel Has Properties Different from
Channels of Other Cell Organelles
The peroxisomal membrane of
leaf peroxisomes contains a channel-forming protein that is different
from those of other cell organelles. The channel shows a single channel
conductance of 350 pS (1 M KCl) which is much lower than those
formed by the porins of mitochondria and chloroplasts under otherwise
identical conditions (see Table 3). Furthermore, the single
channel conductance in salts containing organic anions is much smaller
as expected from the mobility of these ions in the aqueous phase.
Apparently the peroxisomal channel is not a wide, water-filled channel
like the channels of the mitochondrial and the chloroplast porins. The
single channel conductance is not a linear function of the bulk aqueous
concentration, caused either by positive net charges in or near the
channel mouth or by a binding site for anions. This is another
indication that the peroxisomal channel has specialized channel
properties. The size of the peroxisomal channel is somewhat difficult
to obtain from the single channel measurements since only the size of
wide, water-filled channels may be obtained from their
conductance(6, 7) . As shown in Table 2the
K salts of formiate, acetate, glycerate, and glycolate
are able to penetrate the channels, but the channel conductance with
these ions is much smaller than with KCl. It appears from this result
that the channels are just large enough to let these organic anions
pass through. From the size of the permeating molecules, a channel
diameter of about 1 nm may be estimated. The channel appears to be well
suited to enable the transfer of metabolites in and out of the
peroxisomes during photorespiratory metabolism.
Do Other Peroxisomal Membranes Also Contain an
Ion-permeable Channel?
Our data strongly suggest that plant
peroxisomes contain an ion-permeable channel. The question arises
whether pores are a common principle of the permeability properties of
peroxisomal membranes. We mentioned in the Introduction that
peroxisomes from yeast and liver may contain pores that have very
similar properties to those in the outer membrane of the
mitochondria(11, 12, 15) . In particular, the
channels reconstituted from liver peroxisomes show the same high
voltage dependence, commencing at about 20-30 mV, as
mitochondrial porins(7, 11) . We did not observe any
voltage dependence up to 70 mV, indicating that the channel from leaf
peroxisomes described here is not voltage regulated within the
physiological range of membrane potentials. Obviously the channels
attributed to liver peroxisomes have completely different properties
than the channel described in our study. It remains to be studied
whether the relatively small peroxisomal pores characterized here to be
completely different from mitochondrial and chloroplast pores are a
special feature of leaf peroxisomes, or whether they are also present
in peroxisomes from other cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.