(Received for publication, May 31, 1995)
From the
Heme lyases are components of the mitochondrial intermembrane
space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are
synthesized in the cytosol without the typical N-terminal mitochondrial
targeting signal. Here, we have analyzed the mode of import and folding
of the two heme lyases of the yeast Saccharomyces cerevisiae,
namely of cytochrome c heme lyase and of cytochrome c heme lyase. For transport into mitochondria,
both proteins use the general protein import machinery of the outer
membrane. Import occurred independently of a membrane potential,
, across the inner membrane and ATP in the matrix space,
suggesting that the inner membrane is not required for transport along
this direct sorting pathway. The presence of a large folded domain in
heme lyases was utilized to study their folding in the intermembrane
space. Formation of this domain occurred at the same rate as import,
indicating that heme lyases fold either during or immediately after
their transfer across the membrane. Folding was not affected by
depletion of ATP and
or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve
homologs of known folding factors (like Hsp60 and Hsp70). The energy
derived from folding cannot be regarded as a major driving force for
import, since the folded domain could be imported into mitochondria
with the same efficiency as the intact protein. We conclude that
protein folding in the intermembrane space obeys principles different
from those established for other subcellular compartments.
Heme lyases are involved in the biogenesis of c-type
cytochromes and are thought to catalyze the covalent attachment of heme
to the apoforms of the cytochromes(1, 2, 3) .
At present, the genes of three enzymes have been identified, the
cytochrome c heme lyases (CCHL) ()of Neurospora
crassa(4) and the yeast Saccharomyces cerevisiae(5) as well as cytochrome c
heme lyase
(CC
HL) (6) of yeast. These proteins are homologous
and share about 35% amino acid sequence identity (50% similarity). They
are located in the mitochondrial intermembrane space, where they are
peripherally associated with the inner
membrane(7, 8) . The functional role of heme lyases is
only partially understood. CCHL appears to have a dual function during
the biogenesis of cytochrome c. First, it has been
demonstrated to serve as a high affinity binding site for apocytochrome c(7, 9) . By forming a stable complex, CCHL
renders the reversible passage of apocytochrome c across the
mitochondrial outer membrane unidirectional (10) . Second, CCHL
is required for the covalent attachment of heme to apocytochrome c(5, 7) . In this reaction, the two vinyl
groups of heme are linked with cysteines 14 and 17 of apocytochrome c to form thioether bonds. Apart from the fact that the
reduced form of heme is required for the conversion to holocytochrome c(11) , the molecular mechanisms underlying this
complicated reaction are still enigmatic. Even less is known about the
involvement of CC
HL in heme attachment to cytochrome c
in the intermembrane space. A direct function
for CC
HL has been inferred from studies showing heme
addition as a prerequisite for the maturation of the intermediate form
of cytochrome c
(6, 12, 13, 14) .
Unlike most other mitochondrial precursor proteins, heme lyases do
not contain typical mitochondrial targeting sequences (presequences) at
their N termini. Thus, their biogenesis is expected to be distinct from
that of the bulk of mitochondrial preproteins. The import pathway has
been investigated in some detail for CCHL from N. crassa(15) . Its precursor is transported to the functional
location, the outer face of the inner membrane, ()by direct
passage across the mitochondrial outer membrane without a requirement
for the inner membrane (see also (16) ). Import occurs
independently of the inner membrane potential,
, and does
not require external energy sources like ATP. Thus, this pathway is
markedly different from that of other constituents of the intermembrane
space, e.g. the cytochromes c
and b
, which require an energized inner membrane for
import(12) .
Many important aspects of the biogenesis of
heme lyases are not understood. For example, it is unknown whether the
pathway defined by N. crassa CCHL is also shared by the
homologous CCHL and possibly by other preproteins. The
independence of the import on external energy sources like ATP poses
the problem of the driving force of this reaction. The absence of an
N-terminal presequence raises the important question of the location
and nature of the targeting signal in heme lyases. Finally, it is not
known how heme lyases become folded in the intermembrane space and
whether the free energy change of folding can be regarded as a driving
force for import. At present, no information on the mechanism of
protein folding in this compartment is available.
To address some of these questions, we decided to investigate the biogenesis of the two heme lyases from the yeast S. cerevisiae. First, we have elucidated the import pathway of these proteins into their functional location. According to our results, yeast heme lyases are imported directly across the outer membrane with no apparent requirement for the inner membrane. This makes it likely that other proteins of this compartment use a similar sorting pathway. Second, we examined the folding of these enzymes into their native structure by following the generation of a protease-resistant domain upon import into the intermembrane space. Folding was found to occur during or immediately after import. It did not involve folding factors acting similarly to those known from other cellular subcompartments, e.g. Hsp60 and Hsp70 chaperone proteins (17) or peptidylprolyl cis-trans isomerases. Thus, folding in the intermembrane space either occurs in a spontaneous fashion without the assistance of chaperone proteins or folding is mediated by so far unknown factors and mechanisms. Finally, our studies render folding of the heme lyases as a driving force for their import unlikely.
Figure 1:
Import of yeast heme lyases into
isolated mitochondria. A, reticulocyte lysate containing
[S]methionine-labeled CCHL and CC
HL
was mixed with mitochondria in import buffer and incubated for 30 min
at 25 °C. After reisolation of the mitochondria, samples were
treated with the indicated amounts of proteinase K (PK) for 30
min at 0 °C in the presence or absence of 0.1% Triton X-100
detergent as indicated. Proteins were precipitated with trichloroacetic
acid, subjected to SDS-PAGE, and analyzed for imported CCHL and
CC
HL by fluorography. The standard lane (St.)
contains 10% of added heme lyase precursors. B, time and
temperature dependences of the import were analyzed by incubating
mitochondria with CCHL and CC
HL precursors at 0, 10, or 25
°C for the indicated times. After the import, samples were chilled
on ice, and half of each sample was treated with 100 µg/ml
proteinase K for 30 min at 0 °C, whereas the other half remained on
ice. Mitochondria were reisolated by centrifugation, and imported
material was analyzed by SDS-PAGE, fluorography, and densitometry. Data
are given relative to the material bound to mitochondria, which was
measured from the average of material recovered in samples lacking the
protease treatment. C, import of heme lyases is
receptor-dependent. Mitochondria (5 mg/ml) were treated with the
indicated concentrations of trypsin for 15 min at 0 °C in SoH
buffer. Protease activity was halted by addition of 2.5 mg/ml soybean
trypsin inhibitor. After 10-fold dilution with import buffer containing
1 mg/ml soybean trypsin inhibitor, import of the precursors of CCHL and
CC
HL was for 15 min at 25 °C. As a control, the
precursor of the
-subunit of MPP was imported for 5 min at 25
°C. Further analysis for imported proteins was as in B.
The data are given relative to the import without trypsin
pretreatment.
Does the transport
of heme lyases require the protein import complex of the mitochondrial
outer membrane? To examine this question, the protease-sensitive
receptors at the mitochondrial surface were inactivated by pretreating
the mitochondria with trypsin. Import of both heme lyases was strongly
reduced, applying trypsin concentrations known to degrade the surface
receptors (Fig. 1C, higher than 20 µg/ml) (cf.(28) and (29) ). The behavior of a matrix-targeted
protein, the precursor of the -subunit of matrix processing
peptidase (preMPP), was identical. Thus, import of heme lyases into
mitochondria depends on protease-sensitive components of the outer
membrane, suggesting that these proteins use the general protein import
machinery of the mitochondrial outer membrane.
The submitochondrial
localization of imported heme lyases was determined by subfractionation
of the mitochondria. After import, the organelles were treated in
hypotonic buffer to effect swelling of the mitochondria and rupture of
the outer membrane(21) . As a result, proteins of the
intermembrane space and the inner membrane become accessible to
digestion by added protease, while components beyond the inner membrane
remain resistant. Both imported heme lyases behaved identical to the
endogenous enzymes and were degraded after opening of the outer
membrane (Fig. 2A). Thus, they had reached their
functional location, the intermembrane space. Unlike cytochrome b, a soluble constituent of the intermembrane
space, imported and endogenous heme lyases sedimented with the
mitoplasts (i.e. mitochondria with a disrupted outer
membrane), indicating that they were membrane bound. The heme lyases
remained stably associated with the membranes even at higher ionic
strength (Fig. 2B), in contrast to soluble constituents
of the intermembrane space like cytochrome c. To further
analyze the sub-mitochondrial localization of the imported proteins, an
extraction with alkaline buffers was performed(24) . About half
of both endogenous and imported CCHL was recovered in the supernatant,
whereas CC
HL was fully resistant to solubilization (Fig. 2C). Control proteins were recovered in the
supernatant (cytochrome b
or mitochondrial Hsp70)
or in the pellet (ADP/ATP carrier) as previously reported (cf.(30) ). The similar behavior of endogenous and imported
heme lyases suggests the correct localization of the imported material.
Since heme lyases are hydrophilic proteins and do not contain any
obvious membrane-spanning segments, the tight membrane interaction is
unexpected. It remains to be determined which forces anchor heme lyases
to the membrane.
Figure 2:
Endogenous and imported heme lyases are
located in the intermembrane space. A, CCHL and
CCHL precursors were imported into mitochondria for 30 min
at 25 °C. Following proteinase K treatment, mitochondria were
reisolated by centrifugation and resuspended in SoH buffer, and
swelling was performed in the presence or absence of 100 µg/ml
proteinase K. Membranes were reisolated by centrifugation and
resuspended in SoH buffer. Proteins were precipitated with
trichloroacetic acid, and imported material (Imp.) was
analyzed by SDS-PAGE, blotting onto nitrocellulose, and
autoradiography. Endogenous proteins (End.) were analyzed by
immunostaining of the same blot. A controllane shows
the input mitochondria (M.) B, heme lyases are
resistant to salt extraction. Mitochondria were subjected to swelling
reactions to generate mitoplasts. KCl was added from 2-fold
concentrated stock solutions, and samples were incubated for 5 min at 0
°C. After reisolation of the membranes by centrifugation in a
Beckman TLA45 rotor at 224,000
g for 30 min at 2
°C, pellets were resuspended in sample buffer, and supernatant
fractions were precipitated with trichloroacetic acid. Endogenous
proteins were analyzed by SDS-PAGE, blotting onto nitrocellulose, and
immunostaining. Controllanes show mitochondria (M.) subjected to the same reisolation procedure. C,
resistance of heme lyases against alkaline extraction. Following import
and protease treatment as in A, mitochondria were reisolated
by centrifugation, resuspended in 200 µl of freshly prepared 0.1 M Na
CO
, and incubated for 30 min at 0
°C. Half of the sample was precipitated with trichloroacetic acid (Tot.); the other half was centrifuged in a Beckman TLA 45
rotor at 224,000
g for 30 min at 2 °C. The
membrane pellets were resuspended in 0.1 M Na
CO
, and both pellet (Pel.) and
supernatant (Sup.) fractions were precipitated with
trichloroacetic acid. Further analysis for imported or endogenous
proteins was as in A. Cytochrome b
(Cyt. b
) and cytochrome c (Cyt. c) are soluble markers of the
intermembrane space; MIM44 and Hsp70 (heat shock protein of 70 kDa)
are located in the matrix; the ADP/ATP carrier (AAC) is an integral
protein of the inner membrane.
We investigated the energy requirements for the
import of yeast heme lyases. First, the membrane potential,
, was depleted by increasing concentrations of the uncoupler
FCCP. No reduction of import was observed for the two heme lyases, even
at 10-fold higher concentrations of FCCP than those needed to
completely inhibit the import of a matrix-targeted preprotein, preMPP (Fig. 3A). Second, ATP was depleted selectively inside
or outside the mitochondrial inner membrane(27, 31) .
In both cases, the removal of ATP did not cause a significant change of
the import efficiency of the heme lyases (Fig. 3B).
Control proteins such as the
-subunit of F
-ATPase and
precytochrome c
were affected in their import as
reported in previous studies(27, 31, 32) .
Taken together, these import studies show that yeast heme lyases become
imported into the mitochondrial intermembrane space via direct transfer
across the outer membrane by utilizing the general protein import
machinery. Since efficient import can occur in the absence of external
energy sources like a membrane potential,
, or ATP in the
matrix, the inner membrane translocation machinery is apparently not
involved in import.
Figure 3:
Energetics of the import reaction. A, import does not require . Mitochondria in import
buffer were treated with the indicated concentrations of FCCP added
from a 100-fold concentrated stock solution in ethanol for 3 min at 25
°C. Precursor proteins in reticulocyte lysate were added, and
samples were incubated for 20 min at 25 °C (CCHL and
CC
HL) or for 5 min at 25 °C (
-subunit of MPP).
Analysis for imported protein was as in Fig. 1B. The
mature form of MPP was used for quantitation. Data are given relative
to the import without added FCCP. B, import occurs
independently of ATP inside and outside of the mitochondrial inner
membrane. Reticulocyte lysates containing the precursors of either CCHL
and CC
HL or the
-subunit of F
-ATPase (F
) and cytochrome c
(Cyt. c
) were depleted of
ATP by treatment with 100 µg/ml hexokinase/myokinase for 10 min at
25 °C. The depleted lysates were mixed with mitochondria, which had
been selectively depleted of either internal or external ATP (see
``Materials and Methods''), or with control mitochondria,
which had been supplied with an ATP-regenerating system. Samples were
incubated at 25 °C for 15 min (CCHL and CC
HL) or 5 min
(F
-ATPase and cytochrome c
). Further
analysis was as in Fig. 1B. The mature form of
F
-ATPase and the intermediate form of cytochrome c
were used for quantitation. Data are given
relative to the respective import observed with fully energized
mitochondria.
Figure 4:
Heme lyases contain a tightly folded
domain, which is generated upon import. A, purified
mitochondria were reisolated by centrifugation and resuspended at a
protein concentration of 12.5 mg/ml in SoH buffer either lacking or
containing 8 M urea (Step1). Aliquots were
diluted 25-fold into hypotonic buffer (0.06 M sorbitol, 20
mM HEPES, pH 7.4) in the presence of the indicated
concentrations of trypsin and incubated for 20 min at 0 °C (Step2). To control for the effect of residual urea
after dilution, indicated samples contained 0.3 M urea. After
addition of 1 mM PMSF, samples were precipitated with
trichloroacetic acid, and proteins were separated by SDS-PAGE, blotted
onto nitrocellulose, and analyzed for CCHL, CCHL, and their
folded fragments by immunostaining. B, CC
HL was
imported into mitochondria (30 min at 25 °C) by diluting the
precursor 50-fold from reticulocyte lysate (RL) or from 8 M urea into the import mixture. Following proteinase K
treatment, mitochondria were reisolated by centrifugation and
resuspended in SoH buffer, and aliquots were subjected to hypotonic
treatment in the presence of the indicated amounts of trypsin for 20
min at 0 °C. Protease digestion was stopped by addition of 1 mM PMSF, and samples were precipitated with trichloroacetic acid.
Proteins were separated by SDS-PAGE, and imported CC
HL and
its 25-kDa fragment were analyzed by fluorography. As a control,
CC
HL precursor was diluted into SoH buffer lacking
mitochondria (Free). The standard lane (St.) contains
10% of input precursor protein.
We tested whether this fragment was generated in
the course of in vitro import of CCHL. When
CC
HL precursor synthesized in reticulocyte lysate was
imported into mitochondria, the 25-kDa fragment was formed with high
efficiency (more than 50% of intact CC
HL; Fig. 4B). In comparison, only a small amount of
fragment (5-10%) was generated by trypsin treatment of
CC
HL precursor in the absence of mitochondria. When
urea-denatured CC
HL precursor was used in these
experiments, trypsin treatment generated the 25-kDa fragment only
following import into mitochondria. No such fragment was formed upon
dilution of the denatured precursor into buffer lacking mitochondria.
In summary, upon import into mitochondria, CC
HL folds
efficiently into a conformation that is indistinguishable from its
native form. The presence of the 25-kDa folded domain in reticulocyte
lysate, yet in low amounts, indicates that folding is not strictly
dependent on the entry into the intermembrane space. However, the
5-10-fold increase in folding efficiency in the latter case may
indicate the participation of specific factors during the folding
reaction.
The time courses of import and folding were compared. The
formation of the 25-kDa domain proceeded at the same rate as the import
of CCHL, even when the experiments were performed at lower
temperatures to ensure that import occurred in the linear range (Fig. 5). Apparently, folding of CC
HL in the
intermembrane space is at least as rapid as the import reaction, i.e. folding must take place either during or immediately
after the entry of the protein into the intermembrane space.
Figure 5:
Folding of CCHL in the
intermembrane space occurs rapidly. CC
HL was imported into
mitochondria at 0, 12, or 25 °C for the indicated times. Following
proteinase K treatment, mitochondria were reisolated by centrifugation
and resuspended in SoH buffer, and aliquots were subjected to hypotonic
treatment in the absence or presence of 25 µg/ml trypsin for 20 min
at 0 °C. Further analysis was as in Fig. 4B. For
easier comparison of the rates of import and of the formation of the
25-kDa fragment, data are given relative to the respective amounts
obtained at 30 min.
Protein
folding in the mitochondrial matrix or in the cytosol has been reported
to be assisted by heat shock proteins (Hsp60 and Hsp70; e.g. Refs. 25, 33, 34). As these chaperone proteins require ATP for
their function, we asked whether the folding of CCHL would
display a similar dependence on ATP. The formation of the 25-kDa
fragment was analyzed after import of CC
HL in the absence
of ATP. No influence of the presence or absence of the nucleotide on
the amount of fragment formation was observed (Fig. 6).
Likewise, depletion of the membrane potential,
, by the
ionophore valinomycin during import of CC
HL did not alter
the amount of the 25-kDa fragment generated by tryptic digestion (Fig. 6). Therefore, external energy sources do not appear to be
important cofactors in the folding of CC
HL in the
intermembrane space.
Figure 6:
Folding of CCHL occurs
independently of exogenous energy sources and is not impeded by
inhibitors of peptidylprolyl isomerases. CC
HL was imported
into mitochondria for 15 min at 25 °C. Import was performed in the
presence of ATP and NADH (Total), after depletion of ATP by
pretreating mitochondria with oligomycin (20 µM) and
apyrase (40 units/ml) and using reticulocyte lysate, which was
pretreated with apyrase (40 units/ml), after depletion of the membrane
potential,
, by 0.5 µM valinomycin, or in the
presence of either 10 µM cyclosporin A or 10 µM FK506. Further treatments with proteinase K, hypotonic buffer, and
25 µg/ml trypsin were as described in Fig. 4B,
except that apyrase, cyclosporin A, or FK506 were present in the
respective samples at the given concentrations during the reisolation
and swelling procedures. For comparison, CC
HL was diluted
into SoH buffer lacking mitochondria and treated with trypsin as above (free). Data are given relative to the amount of fragment
formed after import into mitochondria in the absence of any
inhibitors.
Heme lyases contain an unusually high number of
proline residues (8-12%). Therefore, the potential role of
mitochondrial peptidylprolyl cis-trans isomerases, namely of
cyclophilin and FK506 binding protein(35, 36) , for
the folding of CCHL was analyzed by using specific
inhibitors of these enzymes(37) . The latter protein has been
found to be localized in the intermembrane space. (
)CC
HL was imported in the presence of 10
µM of either cyclosporin A or FK506, i.e. at
concentrations known to completely inactivate the peptidylprolyl cis-trans isomerase activities(36) . After the import
reactions, the generation of the 25-kDa fragment was tested. No
significant influence of the drugs on the yield of the 25-kDa fragment
was observed (Fig. 6). Even the combined presence of the
inhibitors did not cause any reduction of folding (not shown). Thus,
according to these criteria, neither chaperone proteins, which require
ATP for their function, nor the known mitochondrial peptidylprolyl cis-trans isomerases appear to be involved in the folding of
CC
HL.
Figure 7:
The folded domain of CCHL can
be efficiently imported into mitochondria. A, CC
HL
precursor was treated with the indicated amounts of trypsin for 10 min
at 0 °C in SoH buffer. Then, soybean trypsin inhibitor was added to
a final concentration of 1 mg/ml, and samples were further incubated
for 5 min at 0 °C. Aliquots (10%, Input) were removed and
precipitated with trichloroacetic acid. After addition of 1 volume of
2-fold concentrated import buffer and mitochondria, import was allowed
for 30 min at 25 °C. Further treatment was as in Fig. 1A, except that samples contained 0.5 mg/ml
soybean trypsin inhibitor. Analysis for imported CC
HL or
its 25-kDa fragment was as in Fig. 4B. For easier
comparison of the input and import panels, data were normalized to the
amounts obtained after digestion at 50 µg/ml trypsin. B,
the imported 25-kDa fragment of CC
HL is correctly localized
to the intermembrane space. Generation of the fragment by tryptic
digestion (25 µg/ml) and import was as described in A.
After reisolation, mitochondria were subjected to hypotonic treatment
in the presence or absence of proteinase K (PK) for 20 min at
0 °C. SoH buffer containing 1 mM PMSF was added to a final
volume of 1 ml, and mitochondria were reisolated and precipitated with
trichloroacetic acid. Further analysis of imported CC
HL or
its 25-kDa fragment (Frag.) and of endogenous cytochrome b
(Cyt. b
)
and MIM44 was as in Fig. 2A.
According to our in vitro import studies, yeast heme
lyases reach the intermembrane space by direct transfer across the
outer membrane. The use of the general protein import complex of this
membrane is suggested by the dependence of the import on
protease-sensitive receptors in the same way as found for
matrix-targeted preproteins. The independence of transport from the
inner membrane follows from the energy requirements of the import
reaction. Neither a membrane potential, , across the inner
membrane nor ATP in the matrix space is required for import. Both
conditions are known to be essential for protein transport into and
across the inner
membrane(12, 27, 32, 38) . Thus, the
yeast heme lyases studied here use a direct,
``non-conservative'' import pathway. A similar route has been
deduced recently to be taken by CCHL from N.
crassa(15) .
The import route taken by the three known
heme lyases differs markedly from that of other constituents of the
intermembrane space. In the case of cytochromes c and b
, the involvement of a membrane
potential during import into mitochondria is well
established(12) . At least parts of these preproteins are
transiently exposed to the
matrix(39, 40, 41) , showing that
participation of the inner membrane is a characteristic feature of
their submitochondrial sorting. The pathway defined by apocytochrome c, on the other hand, is similar to that of heme lyases in
that import only requires the outer membrane (for reviews, see Refs. 1
and 3). However, other features of the import of this preprotein are
unique. Transport of apocytochrome c across the outer membrane
occurs independently of protease-sensitive components, in particular of
the general protein import complex(7, 42) .
Nonetheless, specific interaction with this membrane seems to involve a
hitherto uncharacterized component, which also might facilitate the
passage of the apoprotein across the membrane(10) . Import of
apocytochrome c is driven by the specific interaction with
CCHL in the intermembrane space, which serves as a high affinity
``trans side receptor'' before attaching heme to the
apoprotein(7, 8, 9, 10) . Thus,
there exist at least three fundamentally different pathways of protein
sorting into the mitochondrial intermembrane space, one of which is
taken by the heme lyases. These pathways may have been developed as a
result of the evolutionary different origins of the various
intermembrane space proteins. For instance, cytochrome c
, a constituent derived from the bacterial
endosymbiont ancestor, apparently follows a pathway reminescent of
protein export from bacteria (see (40) and (43) ).
Heme lyases, on the other hand, do not appear to have structural
counterparts in bacteria(44) , and therefore may have evolved a
novel way of entering the mitochondrial intermembrane space.
Practically nothing is known about the mechanism of protein folding
in the mitochondrial intermembrane space. To study this process, we
have taken advantage of a large fragment of CCHL that folds
into a trypsin-resistant domain during import. The folded fragment
formed rapidly during or immediately after the import of the protein.
To a low degree, this domain was also generated during translation in
reticulocyte lysate. Therefore, folding does not require the
participation of specific factors from the intermembrane space.
However, the 5-10-fold increase in the efficiency of folding upon
import may indicate assistance by constituents of the intermembrane
space. Even though our study does not identify any factor involved in
the folding reaction, a few components may be excluded. The
independence of folding on the presence of ATP renders it unlikely that
chaperone proteins like those involved in protein folding in, e.g. the cytosol or the mitochondrial matrix, may be involved. The
central components of these folding processes, namely Hsp70 and Hsp60
proteins, need ATP for their function(17) . Furthermore, our
study rules out an essential function of known mitochondrial
peptidylprolyl cis-trans isomerases(35, 36) ,
which in part are localized in the mitochondrial intermembrane
space.
Since all known heme lyases have an exceptionally
high content of proline residues (8-12%), catalysis of their
isomerization might have been expected to accelerate the rate of
folding. Clearly, our studies show that protein folding in the
intermembrane space obeys different principles than those established
for other subcellular compartments.
The mechanism of folding of
CCHL may be a paradigm for other components of the
intermembrane space. A recent study of the folding of other proteins
located in the intermembrane space and of reporter proteins elucidated
remarkable parallels to the folding of heme lyases. (
)Folding occurred rapidly and could not be kinetically
resolved from translocation. There was no apparent requirement for the
presence of external energy sources like ATP. Thus, one might expect
from these studies that for this mitochondrial subcompartment, the
folding process may be assisted by so far unknown chaperone activities.
It will be interesting to identify these components and to further
elucidate the mechanisms underlying the folding reactions.
An open
question concerning the biogenesis of heme lyases has been the nature
of the driving force for their transfer into the intermembrane space.
No external energy sources like cytosolic or mitochondrial ATP were
found to be required for import of all three known heme lyases. As a
possibility for driving the import reaction, the specific folding of
heme lyases in the intermembrane space seems possible(45) .
However, our data do not support this idea; they rather demonstrate
that folding cannot be regarded as the energetic basis for the overall
import process. A domain of CCHL comprising 80% of the
total protein could be efficiently transported into mitochondria
despite its folded character. Therefore, no net energy could be gained
from folding in this case. With the apparent lack of a driving force,
how might the import process be viewed in energetic terms? We would
like to suggest a scenario in which the import is driven by a series of
specific interactions heme lyases undergo during and after import. The
energy derived from these interactions may substitute for the well
studied action of Hsp70 under expense of ATP. A first interaction may
be the recognition of the internal targeting signal of heme lyases by
receptors at the mitochondrial surface. Similar to the case found for
N-terminal presequences, this interaction may be very labile, thereby
facilitating rapid insertion of the polypeptide chain into the
translocation pore(46) . Further movement across the membrane
may then lead to an interaction of the targeting signal with a specific
binding site on the trans side of the outer membrane. If this
interaction is comparatively stable, as has been found with N-terminal
presequences(46) , such a situation would inevitably lead to
net translocation of the targeting signal and regions surrounding the
signal. This would explain why CCHL can be imported into isolated outer
membrane vesicles(16) . Refolding of the translocated
polypeptide chain could then lead to dissociation from the
``trans site'' and thus be prerequisite for
interaction of the heme lyases with the inner membrane. Most likely,
this involves a specific binding partner. The stable interaction with
this putative heme lyase receptor may render the translocation process
irreversible and in addition will ensure the exclusive localization to
the outer face of the inner membrane. These ideas are now open for
direct experimental testing.
It is remarkable that a protein which
is folded before the transport reaction can traverse the outer membrane
with the same efficiency as the unfolded protein. It is well known that
preproteins pass the mitochondrial membranes in an unfolded, extended
conformation(47) . Thus, it seems plausible that also the
soluble, folded 25-kDa domain of CCHL has to undergo an
unfolding step to be able to translocate across the outer membrane. In
general, mitochondrial Hsp70 is believed to participate in such
unfolding reactions(48) , either by actively pulling on the
incoming polypeptide chain (49) or by binding to segments
spontaneously entering the matrix space(50) . In the latter
case, Hsp70 would act as a ``molecular ratchet'' which is
driven by the expense of ATP. Since in the case of CC
HL
precursor Hsp70 molecules are not involved in the import process, the
outer membrane itself may facilitate unfolding. Recently, an example of
such an unfolding reaction has been demonstrated to accompany
initiation of translocation of presequence-containing preproteins
across the isolated outer membrane(46) .