(Received for publication, September 11, 1995; and in revised form, March 12, 1996)
From the
One of the steps in the import of phosphatidylcholine (PC) in
mitochondria is transmembrane movement across the outer membrane. This
process was investigated in vitro using isolated mitochondrial
outer membrane vesicles (OMV) from rat liver. C-Labeled PC
was introduced into the OMV from small unilamellar vesicles by a
PC-specific transfer protein (PCTP). The membrane topology of the newly
introduced PC was determined from its accessibility to phospholipase
A
. Under conditions where the OMV stay intact, externally
added phospholipase A
is able to hydrolyze up to 50% of
both the introduced [
C]PC and the endogenous PC.
Pool size calculations showed that close to 100% of the PC in the OMV
can be exchanged by PCTP. A back-exchange experiment revealed that the
introduction of the labeled PC is reversible. The results demonstrate
that newly introduced PC molecules readily equilibrate over both
leaflets of the OMV membrane. The kinetics of the PCTP-mediated
exchange process indicate that the t
of the
transmembrane movement at 30 °C is 2 min or less.
Phosphatidylcholine (PC) ()is the major constituent
phospholipid in both the inner and the outer membrane of mammalian
mitochondria(1) . The terminal steps of the de novo synthesis of PC (and most other phospholipids) occur primarily on
the cytoplasmic surface of the endoplasmic reticulum(2) .
Consequently, the biogenesis of mitochondria requires efficient import
of PC from the ER. Several mechanisms for the intracellular transport
of phospholipids have been proposed (for a recent review see (3) ). There is growing evidence that the import of
phosphatidylserine into mitochondria occurs via direct interorganelle
contact between mitochondria and the so-called mitochondria-associated
membrane(4, 5, 6, 7) . This ER-like
membrane contains several phosholipid biosynthetic enzymes including
some involved in the synthesis of PC(7, 8) . The
mechanism of PC import into mitochondria is not known.
Additional
mechanisms, so far unknown, are required for the sorting of the newly
imported phospholipids within the mitochondrion, in order to maintain
the specific phospholipid composition and transbilayer phospholipid
distribution of both mitochondrial membranes. In vivo labeling
studies in hepatocytes employing [C]choline have
shown that radiolabeled PC appears rapidly in the mitochondrial outer
membrane, while transfer to the inner membrane is slow(9) .
This suggests that newly synthesized PC is transferred from the ER to
the inner membrane via the outer membrane. In isolated mitochondria,
movement of labeled endogenous PC from the outer to the inner membrane
has been demonstrated(10) . Thus, the outer membrane is the
first barrier to be taken by a newly imported PC molecule on its way to
the inner membrane or to the inner leaflet of the outer membrane.
In
the present study the process of PC movement across the outer
mitochondrial membrane is investigated in an in vitro system
consisting of mitochondrial outer membrane vesicles (OMV) isolated from
rat liver mitochondria. These OMV are sealed and have a right-side-out
topology with an almost symmetric distribution of PC over the inner and
outer membrane leaflet(11) . Phosphatidylcholine-specific
transfer protein (PCTP) was used to introduce radiolabeled PC into the
OMV from donor vesicles. The use of PCTP also allowed the determination
of the pool size of exchangeable PC in the OMV. The transmembrane
distribution of the introduced PC was assessed from its accessibility
to externally added phospholipase A (PLA
). It
is concluded that newly introduced
[
C]dioleoylphosphatidylcholine
([
C]DOPC) equilibrates rapidly over the OMV
membrane with a t
at 30 °C of at most 2 min.
Briefly, the
mitochondria were suspended in hypotonic buffer (5 mM KP, pH 7.2, 5 mM EDTA) at a protein
concentration of 5 mg/ml and incubated on ice for 20 min under
stirring. The outer membrane was detached from the remaining mitoplasts
by 20 strokes in a Potter-Elvehjem homogenizer with a tightly fitting
Teflon pestle. The homogenate (15 ml) was layered on a sucrose step
gradient consisting of 9 ml of 0.25 M sucrose and 12 ml of 1.1 M sucrose in 10 mM MOPS, pH 7.2, 2.5 mM EDTA
(EM buffer). After ultracentrifugation for 1 h at 141,000
g (SW28 rotor, Beckman), the outer membrane fraction was collected
from the 0.25-1.1 M sucrose interface, its sucrose
concentration was adjusted to 1.1 M, and it was loaded on the
bottom of a flotation sucrose gradient (4 ml/tube). A layer of 7 ml
1.065 M sucrose in EM buffer was put on top, and the tube was
filled up with EM buffer. The purified OMV were collected from the
0-1.065 M sucrose interface after ultracentrifugation
for 16 h at 141,000
g (SW41 rotor), diluted in EM
buffer, and pelleted by centrifugation for 1 h at 300,000
g (Ti60 rotor). The pellet was resuspended in 220 mM mannitol, 70 mM sucrose, 2 mM Hepes, pH 7.4 (MSH
buffer) at a protein concentration of 1-3 mg/ml. Aliquots of the
OMV were frozen in liquid nitrogen and stored at -80 °C.
Typically, the procedure yielded 1 mg of OMV/100 mg of mitochondria
based on protein. Based on the total activity of the outer membrane
marker enzyme monoamine oxidase, 25% of the outer membranes are
recovered in the OMV preparation. The purity of the OMV was assessed by
marker enzyme analyses and will be documented in detail elsewhere. ()Typically, the specific activity of monoamine oxidase was
enriched 26 times, while that of succinate cytochrome c reductase (mitochondrial inner membrane marker) was depleted by a
factor of 30, compared with the specific activities determined for the
mitochondria. The specific activities of monoamine oxidase, the
microsomal markers glucose-6-phosphatase and NADPH-cytochrome c reductase, the lysosomal marker acid phosphatase, and the plasma
membrane marker 5`-nucleotidase in the OMV fraction relative to those
of the nuclei-free rat liver homogenate were 69.8, 0.45, 0.55, 2.8, and
1.3, respectively.
Quantification of the transfer of
[C]DOPC to the OMV involved a correction for the
SUV contamination in the OMV pellet using the
H-labeled
nonexchangeable marker. This contamination typically amounted to 10% of
the total amount of SUV added. In the calculation it was assumed that
the
C-specific radioactivity of the co-pelleted SUV is the
same as that determined for the SUV in the supernatant. In order to
calculate the obtained specific radioactivity of PC in the OMV, the
phospholipid phosphorus/protein ratio and the PC content of the OMV
were determined. OMV aliquots corresponding to 150 µg of protein
were extracted according to Bligh and Dyer(16) , and the
phosphorus content of the organic phase was quantitated (17) to
yield the phospholipid phosphorus/protein ratio. Phospholipid analysis
of the OMV extracts by two-dimensional HPTLC was performed as
described(14) . Spots were visualized by iodine vapor and
scraped off, and phospholipid phosphorus was quantitated(17) ,
yielding the PC content of the OMV. Apart from the considerations
above, the calculation of the pool size of exchangeable PC in the OMV
((specific radioactivity of OMV/specific radioactivity of SUV outer
leaflet)
100%) was based on the notion that 65% of the PC in
the donor SUV is accessible to
PCTP(18, 19, 20) .
In back-exchange
experiments, OMV with [C]DOPC introduced as
described above were incubated at 0.25 mg/ml with unlabeled SUV
consisting of DOPC/DOPA (95:5, mol/mol) at a concentration of 1.5
mM based on phosphorus, in the presence of 18 µg/ml PCTP
at 30 °C for the periods of time indicated. OMV were recollected by
centrifugation at 185,000
g as above. Radioactivity in
the pellet and the supernatant was counted, and the percentage of
[
C]DOPC extracted from the OMV was calculated,
taking into account the above corrections, while assuming that 10% of
the SUV with associated
C label co-pellets with the OMV.
Figure 1:
The effect of
PLA treatment on [
C]DOPC introduced
in mitochondrial OMV. [
C]DOPC was transferred
from radiolabeled SUV to OMV by PCTP exchange for 20 min at 30 °C.
OMV were reisolated by centrifugation and incubated at room temperature
with ppPLA
and bvPLA
at a concentration of 0.4
units/ml each in the presence of 0.1 mM Ca
.
At the indicated times, 0.75 mM EGTA was added to inhibit
PLA
action. After extraction, the OMV lipids were analyzed
by HPTLC. A, a radioactivity scan of a TLC plate shows the
distribution of the
C label over DOPC, lyso-PC, and fatty
acid (FA) during the course of treatment with PLA
.
The TLC profile marked excess PLA
was
obtained by incubating the OMV for 10 min with ppPLA
and
bvPLA
at a concentration of 10 units/ml each. CE denotes the position of the
H-labeled nonexchangeable
marker cholesteryl oleoyl ether, which originates from the SUV that
co-pellet with the OMV. The ratio of
[
H]CE/[
C]DOPC (cpm/cpm) in
the donor SUV as detected by the scanner is 1:2.5. B,
quantification of the time-dependent lipolysis of
[
C]DOPC by PLA
shown in A (
), compared with the degradation of the endogenous PC in
OMV under the same conditions (
). The data have not been corrected
for the
C label present in the adhering SUV (see
``Results''). The error bars at t =
10 min represent the standard deviation from four experiments. C, trypsin-resistant adenylate kinase activity remaining after
treatment of the OMV with PLA
for the indicated times as in B. The intactness of the PLA
-treated OMV was
assayed by trypsin digestion of the accessible adenylate kinase. Data
are averages ± S.D. of four
measurements.
After removal of the donor SUV by
centrifugation, the OMV were incubated with a mixture of phospholipases
A from bee venom and porcine pancreas to determine the
membrane topology of the labeled PC molecules. Fig. 1A shows the HPTLC analysis of OMV phospholipid extracts from a
typical experiment. Approximately 40% of the
[
C]DOPC is hydrolyzed within 10 min by the
action of PLA
(Fig. 1, A and B).
Degradation of the remainder proceeds slowly and is accompanied by a
gradual loss of the intactness of the OMV as indicated by the
increasing accessibility of adenylate kinase present in the OMV lumen
to externally added trypsin (Fig. 1C). In the presence
of an excess of PLA
(Fig. 1A), up to 94% of
the [
C]DOPC is degraded in 10 min. This is
accompanied by a loss of barrier function of the OMV membrane as
evidenced by the proteolysis of over 80% of adenylate kinase by trypsin
(data not shown). Under similar conditions with an excess of PLA
present virtually all endogenous PC is hydrolyzed(11) .
The time course of the degradation of [C]DOPC
parallels that of the hydrolysis of endogenous PC under the same
conditions (Fig. 1B). From the amount of
[
H]CE present in the lipid extracts (Fig. 1A) it is estimated that approximately 10% of the
total
C label present originates from co-pelleted SUV.
This contamination may account for the slightly different degrees of
protection from PLA
observed for labeled and endogenous PC
after 10 min of incubation (Fig. 1B). When the time of
the incubation with PCTP is reduced to 3 min, still 60 ± 5% of
the
C label is recovered as intact PC after 10 min of
treatment with PLA
(data not shown). The data suggest that
the newly introduced PC rapidly adopts a transbilayer distribution
similar to that of the endogenous PC, with approximately 50% of the PC
content localized in the inner leaflet and 50% in the outer leaflet in
agreement with (11) .
The existence of a fast
equilibration of newly introduced [C]DOPC across
the OMV membrane is supported by the results depicted in Fig. 2.
The specific radioactivity of the PC pool in the OMV was determined
after different times of exchange by PCTP and compared with that of the
outer leaflet of the donor SUV, as PCTP only has access to the outer
leaflet of phospholipid vesicles(19) . After 5 min of exchange,
the specific radioactivity of both PC pools reaches a similar
equilibrium value (Fig. 2), demonstrating that the entire PC
pool of the OMV has exchanged with the outer leaflet of the SUV. The
exchangeable pool of PC in the OMV was determined in several
independent experiments, yielding an average value of 91 ± 12%
(± S.D., n = 22).
Figure 2:
Phosphatidylcholine exchange by PCTP
between OMV and [C]DOPC-labeled SUV results at
equilibrium in similar specific radioactivity values for the PC pool of
the OMV and the PC present in the outer leaflet of the SUV. OMV at a
concentration of 0.25 mg/ml were incubated with PCTP (18 µg/ml) and
radiolabeled SUV (0.15 mM PC) at 30 °C. At the indicated
time points OMV and SUV were separated by centrifugation, and the
radioactivity in pellet and supernatant was determined. The specific
radioactivity of PC in the OMV (
) and in the outer leaflet of the
SUV (
) was calculated as described under ``Experimental
Procedures.''
After introduction into the
OMV membrane, [C]DOPC is completely extractable
in a back-exchange experiment as shown in Fig. 3. OMV with
[
C]DOPC incorporated were incubated with an
excess of unlabeled SUV (PC
/PC
molar ratio
of 10) and PCTP. Under these conditions all of the
[
C]DOPC is in exchange with the PC in the outer
leaflet of the SUV (Fig. 3), demonstrating the reversibility of
the transmembrane movement.
Figure 3:
The transfer by PCTP of
[C]DOPC from donor vesicles to OMV is
reversible. Shown is the back-exchange experiment, in which
[
C]DOPC-labeled OMV (0.25 mg protein/ml) were
incubated with an excess of unlabeled SUV (1.5 mM P
) and PCTP (18 µg/ml) at 30 °C for different
times. After centrifugation, the radioactivity in the pellet (OMV) and
the supernatant (SUV) was determined, and the amount of
[
C]DOPC transferred from the OMV was calculated.
The data are presented as percentage of the maximum amount of
extractable [
C]DOPC expected at equilibrium, i.e. when the specific radioactivities of OMV and the outer
leaflet of the SUV have reached the same
value.
The present study demonstrates that
[C]DOPC introduced into the outer leaflet of
isolated mitochondrial outer membrane vesicles by PCTP is rapidly
equilibrated over the two membrane leaflets. This conclusion is based
on two lines of experimental evidence. (i) Similar to the extent of
hydrolysis of endogenous PC, only 50% of the introduced
[
C]DOPC can be degraded by externally added
PLA
, while the OMV stay intact, indicating that the other
50% has flipped to the inner leaflet. (ii) The pool size of
exchangeable PC in the OMV is close to 100%. This result, combined with
the reversibility of the exchange process (Fig. 3), indicates
that the probe molecule [
C]DOPC behaves like
endogenous PC. The monophasic kinetics observed for the PCTP-catalyzed
PC exchange process (Fig. 2) do not allow distinction between
the rates of the actual exchange and the PC transmembrane movement.
Consequently, the rate constant of the transmembrane movement of PC
(flip-flop) is close to or higher than the rate constant of the overall
exchange process, which implies that at 30 °C the t
for PC transmembrane movement in OMV is 2 min
or less.
Direct access of PCTP to the inner leaflet of the OMV membrane by permeation could in principle also explain the results obtained in the present study. However, this possibility is considered remote in view of the molecular size of PCTP (25 kDa(26) ) relative to that of cytochrome c (12 kDa), for which the outer membrane is impermeable(27) . Accordingly, labeled PC is not recovered in the inner membrane fraction after its introduction into intact mitochondria by PCTP(20) .
It may seem paradoxical
that on the one hand almost 100% of the PC pool of the OMV is
accessible to PCTP, while on the other hand only half of the introduced
[C]DOPC can be degraded by external PLA
in intact OMV (Fig. 1). A possible explanation would be
that the PCTP-catalyzed exchange process, or PCTP itself, induces the
rapid transmembrane movement of PC in OMV. This possibility cannot be
excluded; however, so far there are no indications that PCTP would have
this ability in other biomembranes (23, 24, 28) . Furthermore, PCTP does not
induce phospholipid transbilayer movement in protein-free phospholipid
vesicles(17, 18) . The alternative explanation, i.e. that the lyso-PC molecules produced by PLA
do
not participate in the transmembrane equilibration of PC, is considered
more likely.
The microsomal contamination present in the
preparations of OMV requires consideration in view of the rapid rate of
PC flip-flop occurring in rat liver microsomes (t
2-3 min at 37 °C(17) ) and the proposed
role of a specialized ER fraction (mitochondria-associated membrane) in
phospholipid transport to mitochondria(4, 8) . Based
on the specific activity of the microsomal marker enzyme
glucose-6-phosphatase in OMV preparations and in microsomes isolated
according to (14) , the microsomal contamination of the OMV
does not exceed 9.4% based on protein.
Given the
phospholipid:protein ratio of microsomes(1) , it can be
calculated that this percentage corresponds to a contamination of at
most 4% on a phospholipid basis. Consequently, microsomal PC hardly
contributes to the total PC pool. Vance (8) has shown that the
specific activity of glucose-6-phosphatase is higher in
mitochondria-associated membrane than in microsomes, whereas that of
NADPH-cytochrome c reductase is lower. There are no
indications that the microsomal fraction contaminating the OMV has
characteristics of mitochondria-associated membrane, as the specific
activities of both enzymes in the OMV relative to those in the rat
liver homogenate are similar (0.45 and 0.55, respectively).
In vitro studies on the import of PC by rat liver
mitochondria have shown that labeled PC is confined to the outer
membrane after its introduction by PCTP from phospholipid
vesicles(11, 20) . These results raised the question
of which of the putative consecutive steps in intramitochondrial PC
transport is limiting, the movement across the outer membrane or the
transfer from outer to inner membrane. The present study strongly
argues that the first step is not limiting. Calculations of the pool
size of exchangeable PC in the outer membrane of intact mitochondria (20) suggest that the occurrence of transmembrane equilibration
in the outer membrane depends on the acyl chain composition and the
type of label of the PC molecule. In the case of
[C]DOPC the size of the exchangeable PC pool in
the outer membrane of intact mitochondria was found to be 56%,
indicating that the label stays in the outer leaflet(11) .
Combined with the present results this suggests that the inner membrane
might have a regulatory role in the transmembrane movement of PC across
the outer membrane.
In a study on the topology of phospholipids in
mitochondrial OMV from Saccharomyces cerevisiae, the
transmembrane movement of PC in the outer membrane was reported to
proceed relatively slowly (t
50
min(29) ). These authors used a different, non-PC-specific
transfer protein and a different donor-acceptor system, which
complicates direct comparison with the present data.
In vivo labeling in rat hepatocytes has shown that 15 min after the
addition of [C]choline, the specific
radioactivity in the mitochondrial outer membrane is approaching
equilibrium with that in the microsomes, which is consistent with a
rapid transmembrane movement of PC in the outer membrane(9) .
The existence of rapid PC transmembrane movement across the mitochondrial outer membrane makes a lot of sense from the point of view of biology. Since mitochondria are highly dynamic organelles that should have easy access to newly synthesized membrane components, it does not come as a surprise that the outer membrane has a mechanism for rapid shuttling of phospholipids. Comparable fast rates of energy-independent phospholipid transmembrane movement have been reported in membranes that are sites of phospholipid synthesis, the ER membrane (18, 30) and the inner membrane of Escherichia coli(31) .