From the
Apocytochrome c follows a unique pathway into
mitochondria. Import does not require the general protein translocation
machinery, protease-sensitive components of the outer membrane, or a
membrane potential across the inner membrane. We investigated the
membrane binding and translocation steps of the import reaction using
purified outer membrane vesicles (OMV) from Neurospora crassa mitochondria. OMV specifically bound, but did not import
apocytochrome c. If, however, specific antibodies were
enclosed inside OMV, apocytochrome c was accumulated in
soluble form in the lumen. Import was reversible, since apocytochrome
c became accessible to external protease after release from
the antibodies. Thus, OMV are competent of translocating apocytochrome
c into their lumen, but lack a binding partner which traps the
apoprotein. In intact mitochondria, cytochrome c heme lyase
(CCHL), a peripheral protein of the inner membrane, serves such a
function by stably associating with apocytochrome c in a
complex which is detectable by co-immunoprecipitation. We suggest a
model for the import mechanism of apocytochrome c in which the
apoprotein specifically associates with and reversibly passes across
the outer membrane. Translocation is rendered unidirectional by stable
association with CCHL which serves as a ``trans side
receptor.'' Finally, heme is attached by CCHL and the holoprotein
folds into its native structure.
Mitochondrial cytochrome c is a soluble protein of the
intermembrane space which participates in the electron transfer between
complex III and complex IV of the respiratory chain (for reviews, see
Refs. 1, 2). As with most of the mitochondrial proteins, cytochrome
c is encoded by a nuclear gene, synthesized on cytoplasmic
ribosomes, and translocated into the organelle in a post-translational
fashion (for reviews, see Refs. 3-5). In the intermembrane space
the apoform of the protein is converted to holocytochrome c by
the enzymatic action of cytochrome c heme lyase
(CCHL)
The
mode of how apocytochrome c passes across the mitochondrial
outer membrane defines a unique import pathway that differs in a number
of criteria from that of other mitochondrial preproteins (for reviews,
see Refs. 10, 11). Apocytochrome c does not carry a cleavable,
N-terminal targeting sequence. Instead, two internal segments of the
apoprotein have been reported to be important determinants for
targeting
(12, 13, 14) . Unlike for most other
mitochondrial preproteins an electrochemical potential across the
mitochondrial inner membrane is not needed for transport
(15) .
Protease-sensitive outer membrane proteins, in particular components of
the mitochondrial receptor complex, do not appear to be required for
import
(6, 16) . Little is known, however, about the
mechanism of passage across the outer membrane. Extensive studies using
artificial liposomes demonstrated a unique membrane insertion activity
of apocytochrome c which is dependent on the presence of
negatively charged phospholipids (e.g. phosphatidylserine; for
review, see Ref. 17). At least parts of the apoprotein are able to
penetrate the lipid bilayer and thus become accessible to proteases
added on the trans side of the membrane
(18) . Based on
these results the spontaneous membrane insertion activity was suggested
to be an important feature of the membrane passage of apocytochrome
c. However, it remained unclear from these studies, whether in
mitochondria apocytochrome c passes across the lipid phase of
the outer membrane, or whether yet unknown, protease-resistant membrane
proteins assist in the transport reaction.
In addition to its
enzymatic function during heme attachment, further involvements of CCHL
in the biogenesis of apocytochrome c were suggested. For
instance, it was proposed that CCHL serves as a high affinity binding
site for apocytochrome c(6, 7) . Mutant
mitochondria lacking CCHL showed only weak
binding
(7, 19) . In an in vivo study
accumulation of apocytochrome c in mitochondria correlated
well with different levels of CCHL expression
(20) . However, a
complex between CCHL and apocytochrome c has not been
demonstrated directly. Thus, the function of CCHL as a ``trans side receptor'' for apocytochrome c remains to be
proven. A possible third role of CCHL as a component assisting in the
membrane passage of apocytochrome c remained elusive from
previous work. In this function, CCHL was suggested to associate with
apocytochrome c, while the latter is still penetrating the
outer membrane
(6) . This might trigger the entry of the
apoprotein into the intermembrane space, e.g. by induction of
a conformational change and/or by enzymatic conversion to the
holoprotein
(21) .
In this paper we have studied the import
mechanism of apocytochrome c. For the mechanistic analysis of
the membrane binding and translocation reactions, we took advantage of
a protein translocation system with isolated outer membrane vesicles
(OMV) from Neurospora crassa mitochondria
(22, 23) . Since these OMV are devoid
of CCHL, it was possible to investigate the outer membrane interaction
of apocytochrome c without interference by this enzyme. We
find that OMV specifically bound apocytochrome c, but did not
import it, unless a high affinity binding partner was present on the
trans side of the membrane. Since specific antibodies
introduced into the lumen of the OMV were sufficient to support import,
we conclude that CCHL is not required for outer membrane passage of
apocytochrome c. Yet, the protein is needed for stable
accumulation of apocytochrome c in the intermembrane space.
Binding to CCHL may therefore provide the major driving force rendering
the reversible translocation process unidirectional.
Based on previous models, a likely binding site
for apocytochrome c was CCHL, which is located at the outer
face of the inner membrane (data not shown, 20). We tried to directly
demonstrate the interaction of apocytochrome c and CCHL in
isolated mitochondria by co-immunoprecipitation experiments.
Mitochondria-bound apocytochrome c was precipitated with
antibodies specific for CCHL, but not by preimmune serum
(Fig. 2). Precipitation was strongly decreased by including KCl
in the binding reaction, a condition known to affect the binding of
apocytochrome c to mitochondria (6, see below). Furthermore,
much less material was co-immunoprecipitated if reduced heme was added
to convert bound apocytochrome c to the
holoprotein
(6) . This results in the release of folded and
soluble holocytochrome c into the intermembrane space. We
conclude that apocytochrome c becomes efficiently imported
into isolated mitochondria where it is found in a complex with CCHL. In
contrast, OMV which are depleted in CCHL more than 600-fold
(Fig. 1D) do not accumulate the apoprotein in their
lumen, even though they are able to bind a substantial fraction.
We have analyzed the membrane binding and translocation steps
of apocytochrome c import into the mitochondrial intermembrane
space. Together with previously obtained results our data lead to a
model for the biogenesis of cytochrome c (Fig. 10). The
precursor, apocytochrome c, is synthesized on cytosolic
ribosomes without a cleavable mitochondrial targeting sequence at its N
terminus. Targeting to mitochondria occurs in a post-translational
fashion. Initial binding to the outer membrane surface may occur via
electrostatic association with phospholipid headgroups and is followed
by specific interaction with a protease-resistant component (
Despite being essential for in vivo and in vitro accumulation of apocytochrome c in the intermembrane
space, CCHL does not appear to be required for the actual membrane
translocation step. Transport across the mitochondrial outer membrane
is observed if apocytochrome c-specific binding partners, like
IgG, are present on the intermembrane space side. Without such a
``trap'' the apoprotein would leave the OMV in a retrograde
translocation reaction. It seems unlikely to us that IgG functionally
participates in the release of apocytochrome c from the outer
membrane and thereby mimicks a possible function of CCHL in
facilitating entry of the apoprotein into the intermembrane space.
Thus, the outer membrane alone can accomplish full translocation of
apocytochrome c, yet a binding site on the trans side
of the membrane is required to shift the equilibrium of the import
reaction. The question, when precisely CCHL starts to interact with
translocating apocytochrome c, is difficult to answer. This
might occur soon after the first appearance of segments of
apocytochrome c in the intermembrane space. An early
interaction of apocytochrome c with CCHL would fit well to a
study in which holo conversion of membrane spanning apocytochrome c fusion proteins was observed
(33) . However, the low
efficiency of heme attachment in these experiments may indicate that
this pathway is not the preferred one in the physiological situation.
As an alternative, apocytochrome c might diffuse through the
intermembrane space as a soluble intermediate before associating with
CCHL. In this view, the function of CCHL is restricted to providing a
binding site for apocytochrome c in the target compartment.
Despite the fundamental differences between the translocation of
apocytochrome c and the general import routes used by other
mitochondrial preproteins there are common principles underlying both
processes. A reversible membrane passage is preceded and followed by
steps introducing both specificity and unidirectionality to the
reaction. Correct targeting is usually verified by the specific
interaction of preproteins with components on the cis side of
the membrane, e.g. targeting factors or surface receptors. In
the case of apocytochrome c, the accuracy of targeting to the
mitochondrial intermembrane space appears to be guaranteed by two
sequential steps, namely by binding to the outer membrane and to CCHL.
Reversibility of membrane translocation has been reported for a number
of experimental systems, e.g. the bacterial inner
membrane
(34) , the membrane of the endoplasmic
reticulum
(35) , and the mitochondrial outer
(23) and
inner membranes (36). In comparison to these transport reactions, the
system described here is remarkable in that the whole preprotein and
not only segments thereof can undergo a retrograde translocation. This
implies that apocytochrome c either passes through the lipid
bilayer or uses a pore-like structure which can be entered and
traversed from both sides.
Reversible membrane transit of
preproteins allows for interaction with components on the trans side of the membrane. In most cases, this can be considered as the
driving force for the whole reaction. For these interactions diverse
mechanisms are used in the various membrane systems. For instance,
repetitive cycles of binding to members of the Hsp70 family and ATP
hydrolysis result in net movement across the endoplasmic reticulum and
mitochondrial inner membranes
(37, 38) . In case of the
mitochondrial outer membrane, the N-terminal presequence, after passing
across a putative translocation pore, associates with a specific
binding site on the intermembrane space side of the membrane (23). For
apocytochrome c, two reactions may be considered to serve as
the driving force for import into mitochondria. First, the interaction
with CCHL, and second, the conversion to the holoform, a reaction also
catalyzed by CCHL. Heme attachment assures dissociation of
holocytochrome c from CCHL. This presumably is triggered by
folding into the native structure. In summary, despite taking a unique
import pathway, apocytochrome c obeys general principles
elucidated for other membrane translocation systems.
We thank Petra Heckmeyer and Marlies Braun for expert
technical assistance and Dr. M. Müller for a gift of E. coli inner membranes. We are grateful to Dr. C. Hergersberg for his
important role in starting these studies and for supplying antibodies
specific for apocytochrome c.
Addendum-When large unilamellar vesicles prepared by the
extruder technique (LUVETs) were used for lipid binding studies, the
results of apocytochrome c binding were comparable to those
presented (R. Baardman and A. Mayer, unpublished results). However, at
higher concentrations of liposomes substantial amounts of apocytochrome
c became bound to liposomes. These data support the conclusion
drawn from the result of Fig. 9, and furthermore provide an
explanation for the differences to previous lipid binding studies (as
summarized by De Kruijff et al., 17).
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(6, 7) , a peripheral
protein of the mitochondrial inner membrane. In this reaction a reduced
heme group becomes covalently linked via two thioether bonds to
cysteines 14 and 17 of the apoprotein
(8, 9) .
Biochemical Procedures
The following published
procedures were used: isolation of mitochondria from N.
crassa strain 74A
(24) , purification of
OMV
(22) , transcription and translation reactions using
reticulocyte lysate (Promega) and [S]methionine
(ICN Radiochemicals) as radioactive label (25), preparation of
C-labeled apocytochrome c by reductive
methylation
(26) , and SDS-polyacrylamide gel electrophoresis
(PAGE) and autoradiography or fluorography of the resulting
gels
(8) . Blotting of proteins onto nitrocellulose and
immunostaining of blotted proteins using the ECL chemiluminescence
detection system (Amersham) was according to the instructions of the
supplier. The resulting films and those from fluorography of
radioactive proteins were scanned on an Image Master densitometer
(Pharmacia). Antisera were raised and immunoglobulin G (IgG) were
purified as described
(27) . IgG were concentrated by
ultrafiltration in Centriprep tubes (Amicon). Protein concentrations
were determined by the Coomassie dye binding assay (Bio-Rad).
Inclusion of Purified IgG into the Lumen of
OMV
OMV (80 µg/ml) were diluted 3-fold with buffer EM (1
mM EDTA, 10 mM MOPS-KOH, pH 7.2) and pelleted for 30
min at 220,000 g in a TLA 100.3 rotor (Beckman). The
OMV were resuspended in 1 ml of EM buffer and spun again for 15 min at
220,000
g to completely remove residual sucrose. After
resuspending the pellet in inclusion buffer (IB; 5 mg/ml BSA, 10
mM MOPS-KOH, pH 6.5) at a protein concentration of 2-4
mg/ml, one volume of purified IgG in water was added (final
concentration 5 mg/ml). The sample was immediately frozen in liquid
nitrogen and then put into a metal block in an ice/water bath to permit
slow thawing. This usually took 15-30 min. After addition of
one-fifth volume of 100 mM MOPS-KOH, pH 7.5, the sample was
incubated for 5 min at 25 °C and adjusted to a sucrose
concentration of 45% by addition of 60% (w/v) sucrose in buffer EMK (EM
plus 150 mM KCl). OMV were recovered from this mixture by
flotation centrifugation (45 min, 140,000
g) through a
gradient consisting of 45%, 40% (both in EMK), 32%, and 8% (both in EM)
sucrose steps (500 µl/step) in a Beckman SW60 rotor. The OMV were
harvested from the 32%/8% sucrose interphase by careful aspiration with
a pipette tip and diluted to the desired protein concentration with SEM
buffer (250 mM sucrose in EM buffer).
Import of Apocytochrome c into Mitochondria and
OMV
S-Labeled apocytochrome c was
synthesized in the reticulocyte lysate, precipitated with ammonium
sulfate (66% saturation), and redissolved in the same volume of SEM
buffer. This treatment removes most of the hemoglobin which interferes
with the electrophoretic analysis of the supernatants of the
translocation experiments. For import 5 µl of this solution were
incubated with mitochondria (100 µg/sample) or OMV (7.5
µg/sample) for 5 min at 25 °C in SEM buffer in a total volume
of 150 µl. Import reactions were chilled on ice, and proteinase K
was added to a final concentration of 30 µg/ml. Protease digestion
was stopped after 15 min at 0 °C by the addition of 2 mM
PMSF and further incubation at 25 °C for 5 min. Mitochondria or OMV
were reisolated (10 min at 15,000
g or 45 min at
125,000
g, respectively) and subjected to SDS-PAGE and
fluorography or autoradiography. In binding experiments where treatment
with proteinase K after the import incubation was omitted, all tubes
were coated with fatty acid-free BSA (1 mg/ml, 15 min) before use in
order to reduce unspecific interaction of apocytochrome c with
tube walls. Pelleted OMV were spun again (5 min, 30,000
g) to remove residual liquid from the tube walls. Finally, the
OMV were resuspended in 20 µl of SEM buffer and transferred to new
tubes where they were dissolved in sample buffer.
Digitonin Fractionation and Sonication of
OMV
After the import reactions, digitonin was added from a 1%
(w/v) stock solution in SEM buffer, and the samples were incubated for
2 min at 0 °C, followed by 20-fold dilution with SEM buffer
containing 100 mM KCl. OMV were reisolated by centrifugation
(45 min, 125,000 g) in a Beckman TLA45 rotor. To the
supernatant 50 µg of BSA or 50 µg of mitochondrial protein was
added as a carrier to quantitatively recover apocytochrome c in the subsequent trichloroacetic acid precipitation. The
trichloroacetic acid precipitates and the reisolated vesicle pellets
were dissolved in sample buffer and analyzed by SDS-PAGE. For
sonication experiments, OMV were reisolated after the import reactions
and resuspended in 600 µl of SEM buffer containing 100 mM
KCl. Samples were sonicated in a glass tube for 1 min at 0 °C
(Branson Sonifier 250 with a microtip, intensity 4, 30% duty cycle).
Membranes and associated material were reisolated and prepared for
SDS-PAGE as described for the digitonin treatment.
Coimmunoprecipitation of Apocytochrome c with
CCHL
IgG were prebound to protein A-Sepharose beads by shaking
25 µl of antiserum specific for CCHL with 10 mg of beads for 1 h in
IP buffer (25 mM potassium phosphate pH 7.2, 100 mM
KCl, and 2% (w/v) octyl glucoside). The Sepharose beads were washed
once with IP buffer and then used for the precipitation. The samples to
be analyzed were diluted 4-fold with IP buffer, incubated for 15 min at
0 °C, and spun for 15 min at 30,000 g. The
supernatant was added to the Sepharose beads with prebound IgG and
shaken gently for 30 min at 4 °C. The beads were washed twice with
1 ml of cold IP buffer and once with cold 25 mM potassium
phosphate, pH 7.2, before resuspending in sample buffer.
Preparation of Liposomes
Lipids (from Sigma) were
mixed in chloroform in the following molar ratios: 33% bovine brain
phosphatidylcholine (P-6638), 30% Escherichia coli phosphatidylethanolamine (P-3511), 7% bovine liver
phosphatidylinositol (P-2517), 4% bovine brain phosphatidylserine
(P-6641), 1% bovine heart cardiolipin (C-1649), 25% ergosterol
(E-6510). The solvent was removed in a rotary evaporator, and the lipid
film was overlaid with EM buffer. Liposomes were formed by adding glass
beads (2 mm diameter), shaking the tube vigorously on a vortex, and by
subsequent sonication (Branson sonifier 250, stage 3, 20% duty cycle).
The liposomes were pelleted by centrifugation (30 min, 125,000
g), resuspended in EM buffer at a lipid concentration of 2
mg/ml and stored at -20 °C. After thawing aliquots were
sonicated briefly before use to disperse aggregates formed during the
freeze-thaw cycle.
Mitochondria, But Not Isolated OMV, Can Accumulate
Apocytochrome c in a Protease-protected Location
Purified OMV
were incubated with S-labeled apocytochrome c. A
considerable fraction of the added apoprotein (40%) was recovered with
the OMV after reisolation by centrifugation, i.e. had bound to
the OMV (Fig. 1A). All bound material was sensitive to
externally added proteinase K and therefore had not become imported
into the lumen of the OMV. The integrity of the outer membrane was
assayed by the accessibility of MOM38 to added protease. This integral
membrane protein is converted to a 26-kDa N-terminal fragment
(MOM38*) by proteinase K digestion from outside, but becomes
further degraded, when the outer membrane is opened (22).
(
)
In isolated OMV MOM38 was quantitatively converted to
MOM38* demonstrating the sealed nature of the OMV, even after the
import reaction (Fig. 1B). Apparently, purified OMV can
bind a substantial fraction of added apocytochrome c, but they
do not accumulate the apoprotein in a protease-protected form.
Figure 1:
Apocytochrome c is
imported into mitochondria but not into purified OMV. A,
incubation of S-labeled apocytochrome c with
mitochondria (Mit) or OMV was for 5 min at 25 °C. The
samples were chilled to 0 °C, treated with the indicated amounts of
proteinase K (15 min, 0 °C). Mitochondria or OMV were reisolated by
centrifugation, and proteins were resolved by SDS-PAGE and blotted onto
nitrocellulose. Imported apocytochrome c was visualized by
autoradiography of the blot and quantitated by densitometry.
B, to control for the sealed nature and for the
right-side-out orientation of the OMV, the accessibility of various
reporter proteins to proteinase K was tested. The blots from A were immunostained using antisera against porin, MOM38, and MOM19.
Quantitation was by densitometry. MOM38* represents the
N-terminal 26-kDa fragment of MOM38 generated by externally added
proteinase K. The signals obtained in samples not treated with
proteinase K were set to 100%. C, the intactness of the outer
membrane of isolated mitochondria was analyzed as in B. In
addition, the blots were immunodecorated with antibodies against CCHL.
D, the more than 600-fold depletion of CCHL in purified OMV
was estimated by subjecting mitochondria (10 µg) and OMV (120
µg) to SDS-PAGE and blotting of proteins onto nitrocellulose. CCHL
was detected by immunostaining and quantitated by densitometry. The
data contain a correction for the outer membrane content of
mitochondria (6%, 22). a.u., arbitrary
units.
Apocytochrome c became bound to isolated mitochondria even
more efficiently (Fig. 1A). Two-thirds of bound
apocytochrome c were resistant to externally added proteinase
K, i.e. had become imported into mitochondria, even though the
experiments were performed under non-reducing conditions which do not
support the formation of the holoprotein
(8, 9) . The
integrity of the outer membrane was determined by using MOM38 and CCHL
as marker proteins. 70% of the mitochondria contained an intact outer
membrane (Fig. 1C). Since also about two-thirds of bound
apocytochrome c were protected against proteolytic attack
(Fig. 1A), we conclude that practically all of the
mitochondria-associated apocytochrome c was bound to a site
beyond the surface of the outer membrane. In an earlier investigation
comparatively small amounts of protease-resistant apocytochrome c (10-15% of input) were observed
(6) . The reason for
this discrepancy most likely is that the outer membrane was opened in
these experiments thus allowing added proteases access to the
intermembrane space.
Figure 2:
Coimmunoprecipitation of apocytochrome
c with CCHL. C-Labeled apocytochrome c was incubated with mitochondria (30 µg/sample) for 5 min at 25
°C in the presence or absence of 100 mM KCl. Mitochondria
were reisolated and resuspended in SEM buffer. Aliquots of the sample
received 1 µM heme and 1 mg/ml sodium dithionite
(DT) or an equivalent amount of SEM buffer. After 3 min at 25
°C, the samples were chilled on ice and diluted 4-fold with IP
buffer. Proteins were subjected to co-immunoprecipitation with
antiserum against CCHL or with preimmune serum. Immunoprecipitated
proteins were analyzed by SDS-PAGE and fluorography. The signal
obtained with antiserum against CCHL in the absence of KCl and heme/DT
was set to 100%. This represents about 15% of apocytochrome c bound to mitochondria.
OMV Containing IgG against Apocytochrome c Accumulate the
Apoprotein in a Protease-resistant, Soluble Form
We addressed
the question whether CCHL plays an active role in assisting outer
membrane passage of apocytochrome c, or whether the enzyme
only passively provides a binding site in the intermembrane space. In
the latter case, it should be possible to replace CCHL by other binding
partners for apocytochrome c in the lumen of the OMV. To this
end, purified immunoglobulin G (IgG) which specifically recognized
apocytochrome c (data not shown) was enclosed inside the OMV
by a recently developed freeze-thaw procedure
(28) . During the
slow thawing period, the membranes are transiently opened allowing
equilibration of the vesicle lumen with the external solution. The
antibodies were recovered with the OMV only, when added before the
freeze-thaw procedure, but not when this procedure was omitted, or when
IgG was added after the freeze-thaw treatment (Fig. 3A).
After the freeze-thaw step, the OMV maintained a right-side-out
orientation as indicated by the protease sensitivity of MOM19. In
addition, most of the OMV were sealed as judged from the conversion of
MOM38 to its 26-kDa fragment, MOM38* (Fig. 3B,
cf. Fig. 1, B and C).
Figure 3:
Purified IgG can be entrapped in the lumen
of OMV by a freeze-thaw treatment. A, OMV were incubated with
purified IgG and subjected to a freeze-thaw treatment. IgG was added
either before (B) or after (A) the freeze-thaw step.
Samples which were not frozen remained on ice. After frozen samples had
been thawed, all samples were incubated on ice for a further 20 min.
OMV were reisolated by flotation centrifugation, and their IgG content
was analyzed by SDS-PAGE, immunostaining, and quantitation by
densitometry. B, the intactness of the membrane was analyzed
with OMV which had been subjected to a freeze-thaw procedure or
mock-treated on ice. OMV were reisolated and resuspended in SEM buffer.
One aliquot was treated with proteinase K (40 µg/ml, 15 min, 0
°C), while another one was left on ice. Proteins in both samples
were precipitated with trichloroacetic acid and subjected to SDS-PAGE
and immunostaining using antibodies raised against MOM19 and an
N-terminal peptide of MOM38. Generation of MOM38* (see Fig.
1B) was used as a marker for sealed nature of the OMV, and the
protease accessibility of MOM19 for their correct orientation (22).
Data are given as the fraction of the signals obtained with proteinase
K-treated and untreated samples.
OMV preloaded
with apocytochrome c-specific IgG were incubated with
apocytochrome c. 15% of the added apoprotein became resistant
to proteinase K, i.e. had completely become transported across
the outer membrane (Fig. 4A). No protease-resistant
material was observed when the freeze-thaw step had been omitted or
when IgG had been added after the freeze-thaw step. Similarly,
enclosure of IgG purified from preimmune serum did not result in
protease resistance of apocytochrome c. In agreement with
earlier import studies using intact mitochondria
(6) , import
into IgG-loaded OMV was unaffected, when surface components of the
outer membrane were degraded by pretreatment with proteinase K
(Fig. 4B). Taken together, our data show that for
accumulation of significant amounts of apocytochrome c a high
affinity binding site inside the lumen of the OMV is both necessary and
sufficient. Apparently, neither protease-sensitive components on the
surface of the outer membrane nor CCHL are essential for the transport
of apocytochrome c across the outer membrane.
Figure 4:
Specific antibodies introduced into the
lumen of OMV can trap apocytochrome c in a protease-protected
location. A, OMV were loaded with purified IgG specific for
apocytochrome c (Apoc) or derived from
preimmune serum (Pre-Imm.) as described in Fig. 3A.
After reisolation the IgG-loaded OMV were used for import assays (5
min, 25 °C) using radiolabeled apocytochrome c synthesized
in reticulocyte lysate. The samples were chilled to 0 °C and
divided in half. One aliquot of each sample was treated with proteinase
K (30 µg/ml, 15 min, 0 °C), the other one was left on ice. OMV
were pelleted by centrifugation and subjected to SDS-PAGE and
fluorography. For comparison a 15% input standard was included on the
gel. B, OMV in EM buffer were treated with the indicated
amounts of proteinase K for 15 min at 0 °C. After stopping
proteolysis by addition of 1 mM PMSF, OMV were reisolated and
resuspended in inclusion buffer. Apocytochrome c-specific IgG
were enclosed and import was performed as described in A.
a.u., arbitrary units.
Previous
studies have shown that import of apocytochrome c into
mitochondria and its interaction with CCHL is abolished at higher salt
concentrations
(6) . When tested in the OMV system, both the
binding to OMV and the import into IgG-loaded OMV were salt-sensitive
(Fig. 5A). The salt sensitivity of the latter reaction
was identical to that reported for intact mitochondria
(Fig. 5B, 6). Inhibition of import at higher salt
concentrations was not a result of an inefficient interaction of
apocytochrome c with the IgG enclosed inside the OMV, since
apocytochrome c could be immunoprecipitated with the same
efficiency under all conditions tested. Thus, in addition to the
salt-sensitive interaction of apocytochrome c with CCHL (see
above) binding to and transfer across the outer membrane appear to be
affected at higher ionic strength. This makes it likely that
apocytochrome c itself is the target of the salt sensitivity.
The apoprotein may be rendered incompetent for both membrane insertion
and CCHL interaction by undergoing a conformational change.
Figure 5:
Binding and import of apocytochrome c are salt-sensitive. A, purified IgG specific for
apocytochrome c were added to the OMV. After a freeze-thaw
step or a corresponding mock treatment on ice, OMV were reisolated by
flotation centrifugation. Import of apocytochrome c into the
OMV was performed in the absence or presence of 300 mM KCl for
5 min at 25 °C. The samples were chilled on ice and divided in
half. One aliquot was treated with proteinase K (30 µg/ml, 15 min,
0 °C) while the other one was left on ice. Then, the OMV were
reisolated and subjected to SDS-PAGE and fluorography. B, to
analyze the influence of KCl on the binding of apocytochrome c to IgG, purified IgG was prebound to protein A-Sepharose beads and
incubated with radiolabeled apocytochrome c in SEM buffer (200
µl total volume) in the presence of increasing amounts of KCl (15
min, 25 °C). The Sepharose beads were recovered by centrifugation
and washed three times with SEM buffer. Finally, the beads were treated
with sample buffer, and solubilized apocytochrome c was
visualized by SDS-PAGE and fluorography. Unspecific binding of
apocytochrome c to protein A-Sepharose alone was negligible
under these conditions (not shown). For direct comparison an import
reaction into IgG-loaded OMV was performed under the same buffer
conditions (for details see Fig. 4). a.u., arbitrary
units.
We
tested whether apocytochrome c translocated into IgG-loaded
OMV was soluble in the lumen or was still associated with the outer
membrane. After import the vesicles were opened by treatment with
increasing amounts of digitonin
(29) . Membranes were reisolated
by centrifugation, and pellets and supernatants were analyzed for the
content of apocytochrome c and IgG. Both proteins were
released efficiently into the supernatant at digitonin concentrations
which are known to selectively disrupt the outer membrane
(Fig. 6A, cf. 22). Membrane proteins were not
solubilized at these concentrations of digitonin, since the amount of
porin in the pellet fractions remained unchanged. The soluble character
of imported apocytochrome c was confirmed in sonication
experiments. The import samples were briefly sonified and membranes
were reisolated by centrifugation. The majority of imported
apocytochrome c and of IgG was recovered in the supernatant,
whereas porin as a control appeared in the pellet fraction
(Fig. 6B). These independent approaches demonstrate that
apocytochrome c became translocated into the lumen of the OMV,
where it was in a soluble complex with IgG.
Figure 6:
Apocytochrome c imported into
IgG-containing OMV is soluble in the lumen. Enclosure of IgG directed
against apocytochrome c (Apoc) and import
of apocytochrome c was performed as in Fig. 4. A,
identical aliquots of the import reaction were extracted with
increasing concentrations of digitonin. After centrifugation the
supernatants (sup) and pellets were analyzed by SDS-PAGE and
blotted onto nitrocellulose. Apocytochrome c was visualized by
autoradiography, whereas the amounts of porin and IgG were estimated by
immunostaining of the same blot. The values obtained for the sample
without digitonin treatment were set to 100%. B, another
aliquot of the import reaction was sonicated, while a second one was
left on ice. The two samples were centrifuged, and pellets (P)
and supernatants (S) were analyzed as described in A.
Data are given relative to the input measured by precipitating a third
aliquot with trichloroacetic acid immediately after the import
reaction.
Translocation of Apocytochrome c across the Outer
Membrane Is Reversible
The results presented above show that
apocytochrome c is accumulated on the intermembrane space side
of the outer membrane, if a high affinity binding partner is available.
This suggested that apocytochrome c can traverse the outer
membrane in a reversible fashion. We sought to directly demonstrate the
reversal of the translocation reaction by disrupting the interaction
between apocytochrome c and IgG with 20 mM
-mercaptoethanol and increasing amounts of urea. At 2 M
urea, 60% of the imported apocytochrome c became accessible to
added proteinase K (Fig. 7A). Release of imported
apocytochrome c from the OMV was fast and occurred at 0
°C. OMV remained sealed as evident from the quantitative generation
of the MOM38 fragment (Fig. 7B). The protease was still
active at higher urea concentrations, since upon opening of the OMV by
sonication or upon the addition of digitonin, MOM38 was completely
degraded. Thus, in OMV imported apocytochrome c can regain
access to the cytosolic face of the outer membrane by a retrograde
translocation reaction.
Figure 7:
Imported apocytochrome c becomes
accessible to external protease after release from IgG. A,
apocytochromec was imported into IgG-loaded OMV as
described in Fig. 4. The sample was split, and aliquots were
supplemented with 20 mM -mercaptoethanol and different
concentrations of urea to disrupt the interaction between apocytochrome
c and IgG. Each aliquot was again split into three portions.
The first one (Intact) was treated with proteinase K (250
µg/ml, 15 min, 0 °C), the second one received the same
treatment in the presence of 0.05% (w/v) digitonin (Dig.), and
the third one was sonicated in the presence of the protease (30 s) and
incubated for further 15 min at 0 °C. After adding 2 mM
PMSF, all samples received 50 µg of BSA and were precipitated with
trichloroacetic acid. Proteins were separated by SDS-PAGE and blotted
onto nitrocellulose. Protease-resistant apocytochrome c was
determined by autoradiography of the blot. B, the intactness
of the OMV at the various urea concentrations used was determined by
immunodecoration of the blot from A using antiserum against
the N-terminal segment of MOM38. MOM38*, 26-kDa fragment of
MOM38.
Reversibility of apocytochrome c import has been reported for intact mitochondria
(30) . This
observation seemed to be in contradiction to other reports of a stable
association of apocytochrome c with mitochondria (see above,
20). It was, however, obtained with a heterologous system which may
explain the labile interaction between CCHL and apocytochrome
c. We therefore examined in our homologous N. crassa system whether, at least in a longer time frame, dissociation from
CCHL and consequently release from intact mitochondria would occur.
Apocytochrome c was incubated with isolated mitochondria and,
after reisolation of the mitochondria, proteinase K was added.
Incubation was continued at 0 or 25 °C to test for the protease
accessibility of imported apocytochrome c. In parallel, the
integrity of the outer membrane was estimated by measuring the
resistance against protease of endogenous CCHL. At 0 °C no
significant decrease of protease-protected apocytochrome c was
observed, even after prolonged times of incubation
(Fig. 8A). Previous studies have shown that even at this
low temperature import is fast and efficient
(6) . In contrast,
at 25 °C apocytochrome c became accessible to proteinase K
with a half-time of 35 min (Fig. 8B). However, this
decrease was paralleled by degradation of endogenous CCHL, and
therefore was due to the opening of the outer membrane rather than the
release of apocytochrome c from the intermembrane space. Thus,
in intact mitochondria the stable interaction of apocytochrome c with CCHL precludes its dissociation from the intermembrane space
and ensures the unidirectionality of the import reaction.
Figure 8:
Imported apocytochrome c does not
dissociate from mitochondria. Radiolabeled apocytochrome c (Apoc) was imported into isolated mitochondria
for 5 min at 0 °C. Mitochondria were reisolated, resuspended in SEM
buffer, and incubated in the presence of 20 µg/ml proteinase K at 0
°C (A) or in the presence of 7 µg/ml proteinase K at 0
°C (B). At the indicated time points, aliquots were
withdrawn and supplemented with one volume of SEM buffer containing 2
mM PMSF. After reisolation the mitochondrial pellets were
analyzed by SDS-PAGE and blotting onto nitrocellulose. Apocytochrome
c was detected by autoradiography, porin and CCHL by
immunostaining. The values obtained for the first aliquot withdrawn
were set to 100%.
Binding of Apocytochrome c to the Mitochondrial Outer
Membrane Is Specific
Finally, we asked whether the interaction
of apocytochrome c with the outer membrane is specific or
would also occur with other biological membranes or liposomes. Compared
to OMV apocytochrome c bound very inefficiently to inverted
vesicles derived from the inner membrane of E. coli or to
liposomes composed of synthetic lipids similar to those present in the
mitochondrial outer membrane (Fig. 9, 31). Similar amounts of
apocytochrome c were found in the pellet fraction of a binding
assay even in the absence of any added membranes. Therefore, we
conclude that apocytochrome c exhibits a high preference for
binding to the mitochondrial outer membrane. This preferential binding
may be important to ensure correct targeting to mitochondria and may,
in addition to the association with CCHL, contribute to the overall
targeting specificity.
Figure 9:
Binding of apocytochrome c to the
mitochondrial outer membrane is specific. Apocytochrome c was
incubated for 5 min at 25 °C with OMV (10 µg protein/sample),
E. coli inverted inner membrane vesicles (20 µg
protein/sample), liposomes (20 µg lipid/sample), or without any
membranes. Binding was measured by centrifugation of the samples (45
min, 125,000 g), and the pelleted fractions were
analyzed by SDS-PAGE and fluorography. The signal for apocytochrome
c bound to OMV was set to 100%.
in
Fig. 10
). The apoprotein then becomes translocated across the
outer membrane, a reaction which apparently can be reversed. Either
during the release from the membrane or after diffusion across the
intermembrane space as a soluble intermediate, apocytochrome c gains access to CCHL, a protein bound to the outer face of the
inner membrane. CCHL serves as a high affinity binding partner on the
trans side of the membrane thereby shifting the equilibrium of
the transport reaction toward the intermembrane space. In addition to
the specific binding to the outer membrane this complex formation with
CCHL may be considered the major contribution to the overall targeting
specificity. In a final step, heme is covalently attached to cysteines
14 and 17. This reaction is catalyzed by CCHL and requires heme in its
reduced state. Folding to native holocytochrome c may then
trigger the release from CCHL thus completing the process.
Figure 10:
Model for the biogenesis of cytochrome
c. denotes a putative component in the outer membrane
(OM) facilitating specific binding and membrane passage of
apocytochrome c. For further explanations see text.
IMS, intermembrane space; IM, inner membrane;
SH, thiol groups of cysteines 14 and
17.
Our
investigations show that apocytochrome c can specifically bind
to the mitochondrial outer membrane. Previous studies using intact
mitochondria have failed to demonstrate this interaction, since
interaction with the outer membrane is only transient, and due to its
high affinity the apoprotein is further transported to CCHL
(6) .
At present it remains elusive what the chemical nature of the component
contributing specificity of binding to the outer membrane might be.
Based on extensive studies with artificial liposomes (for review, see
Ref. 17) lipids may be expected to play an important role in this
process. In particular, acidic phospholipids, e.g. phosphatidylserine, have been demonstrated to be required for an
unusual membrane insertion activity of apocytochrome c.
However, our study failed to demonstrate significant binding of
apocytochrome c to artificial liposomes representing the lipid
composition of the outer membrane, at least under the conditions used
with OMV. Thus, if lipids alone are providing the basis for specific
binding, then such a compound must be a special lipid of the outer
membrane. Alternatively, an unknown proteinaceous component may be
responsible for directing apocytochrome c to the mitochondrial
surface. Such a component might not only assure specific binding of the
apoprotein, but also function in passing it across the membrane. A
recently identified component of cytochrome c biogenesis,
Cyc2p, is an unlikely candidate, since the protein was reported to be
localized to the mitochondrial inner membrane
(5, 32) .
Clearly, direct proof for the existence of such a component is
required. Our studies using isolated OMV pave the way for approaching
this goal by making it possible to reconstitute the binding and import
processes with liposomes and detergent extracts of the outer membrane.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.