From the
Precytochrome b
Cytochromes c
Presently, there are two opinions as to how this
intermembrane space sorting signal operates, namely the ``stop
transfer'' model and the ``conservative sorting'' model
(for review, see Glick et al. (1992b)). The stop transfer
model proposes that the precursors initiate import along the general
import pathway, but the sorting signal serves to arrest them in the
inner membrane import machinery. Completion of translocation across the
outer membrane is proposed to be driven by the dissociation of the
outer and inner membrane import channels. Lateral diffusion out of the
import site together with the maturation by the Imp1p protease in the
case of cytochrome b
An alternative mechanism for the sorting to the
intermembrane space is the conservative sorting model, which accounts
for the resemblance of the intramitochondrial targeting sequence to
prokaryotic leader sequences (Hartl et al., 1986, 1987; Hartl
and Neupert, 1990). This model proposes that the precursors are
imported along the general import pathway. Upon emergence in the
matrix, the sorting signal initiates a retranslocation process back
across the inner membrane to the intermembrane space. According to this
model, import through the matrix could occur concomitantly with
retranslocation back across the inner membrane.
Earlier experimental
data suggesting conservative sorting have been challenged by Glick et al. (1992a). The criticism concentrated on technical
aspects that led, as the authors concluded, to an erroneous
interpretation of the data in favor of conservative sorting. We have
extended our previous investigations to determine as to whether import
intermediates of cytochrome b
Radiolabeled precytochrome b
We then
investigated whether the intermediate-size species, once located in the
matrix, could be chased to the intermembrane space in a second
incubation reaction. For these experiments, the intermediate-size
species was accumulated in the matrix by import for 10 min at 12
°C. Mitochondria were subjected to hypotonic swelling in the
presence of a low concentration of protease (for details, see
``Materials and Methods''). The resulting mitoplasts were
then further incubated at 25 °C in order to allow retranslocation
across the inner membrane into the intermembrane space. However, the
matrix-localized intermediate could be neither chased across the inner
membrane nor converted into the mature-size form, but rather remained
stable in the mitoplasts throughout the chase period (Fig. 2D). Thus, it appears that once stalled, the
export process cannot be reinitiated. As no proteolytic degradation of
this matrix-localized species was observed throughout this prolonged
chase, the decrease in the matrix-located intermediate-size form
observed in the kinetic analysis in intact mitochondria (Fig. 2C) probably reflects further export to the
intermembrane space.
Radiolabeled pb
The data demonstrate that
pb
Sorting of cytochrome b
The sorting kinetics of cytochrome b
In agreement with the data presented
above, the current model of conservative sorting postulates the
following scenario. Sorting to the intermembrane space requires that at
least the complete bipartite presequence of cytochrome b
According to a stop transfer mechanism, inhibition of the first
processing event by matrix processing peptidase should not affect the
transmembrane arrest of cytochrome b
Inhibition of precursor processing was also observed for
precytochrome b
Several amino acid residues in the
sorting sequence have been demonstrated to be crucial for sorting to
the intermembrane space (Beasley et al., 1993; Schwarz et
al., 1993). Both sorting models agree that a very specific
recognition step is required for sorting to the subcompartment. This is
easy to imagine with a conservative sorting mechanism, but poses
problems with a stop transfer process. In the latter, membrane
components, which form the import channel for the passage of hundreds
of matrix-targeted precursors, in the case of cytochrome b
These findings on the signal sequence
requirements imply that a putative stop transfer component located in
the import channel would have to recognize a segment longer than 50
residues. Bearing this in mind, it is hard to conceive how such a long
stretch can operate as a stop transfer signal in the inner membrane
during the translocation process. One would have to postulate that it
must adopt a complex secondary structure during translocation. At the
same time, the inner membrane channel would display an inertness toward
all matrix-targeted proteins and a number of inner membrane proteins
that contain similar hydrophobic stretches and that are clearly sorted
into the matrix (Mahlke et al., 1990; Rojo et al.,
1995). Furthermore, the sorting process was shown to be affected at
reduced temperatures for pb
Finally, in this study,
we have focused on the transient accumulation of intermediate-size
forms of cytochrome b
We thank Gabi Ludwig and Sandra Weinzierl for
excellent technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
is targeted to the
mitochondrial intermembrane space by a dual targeting sequence
comprising 80 amino acids. A kinetic analysis of intramitochondrial
sorting was performed. The intermediate-size form accumulated
transiently in the matrix. When import was performed in the presence of
metal chelators to prevent the first processing by the matrix
processing peptidase, >40% of the imported precursor was localized
in the matrix. A deletion of 13 amino acids in the intermembrane space
sorting sequence caused partial inhibition of the first processing, and
a transient accumulation of the precursor form in the matrix was also
observed. The decrease in this matrix-localized precursor form
paralleled an increase in the mature-size form in the intermembrane
space. A point mutation in the mitochondrial targeting sequence
(N-terminal to the sorting sequence) resulted in missorting to the
matrix space. Furthermore, a chimeric protein consisting of the initial
85 residues of cytochrome b
fused to dihydrofolate
reductase was partially targeted to the matrix at 15 °C, but not at
25 °C. Together, the results presented here indicate that
cytochrome b
passes through the matrix on its
sorting pathway to the intermembrane space.
and b
are nuclear encoded proteins (Guiard, 1985; Sadler et
al., 1984; Römisch et al., 1987). They are
synthesized in the cytosol and are subsequently imported into
mitochondria, where they are located in the intermembrane space. The
precursors of both cytochromes possess cleavable bipartite signal
sequences. The first part of this signal is a mitochondrial targeting
sequence (Hartl et al., 1989) and directs the precursor to the
mitochondria, where it mediates a potential-dependent insertion into
the inner membrane and undergoes cleavage by the matrix processing
peptidase complex in the mitochondrial matrix. The second part
resembles a bacterial leader sequence and directs the intermediate-size
forms into the intermembrane space (Koll et al., 1992; Jensen et al., 1992; Beasley et al., 1993; Schwarz et
al., 1993). The proteins are then finally processed to the
mature-size forms by Imp1p/Imp2p proteases located at the outer face of
the inner membrane (Ohashi et al., 1982; Behrens et
al., 1991; Schneider et al., 1991; Nunnari et
al., 1993).
would result in the release
of soluble cytochrome b
and assembly into
tetrameric complexes in the intermembrane space (Glick et al.,
1993). In the case of cytochrome c
, a C-terminal
anchored inner membrane protein, it is proposed that once imported into
the intermembrane space, the C-terminal segment undergoes a second
independent insertion step into the inner membrane (Wachter et
al., 1992).
pass through the
matrix on their way to the intermembrane space. The data summarized
below indicate that the sorting of cytochrome b
indeed occurs via the matrix. Furthermore, evidence is presented
that retranslocation into the intermembrane space may proceed mainly in
parallel with the import process.
Isolation of Mitochondria
Wild-type Saccharomyces cerevisiae (D273-10B) cells were grown in
lactate medium (Daum et al., 1982). Cells were collected at an A of 1.5, and mitochondria were isolated
according to Daum et al.(1982).
Protein Import into Isolated Mitochondria
Import
of radiolabeled precursor into isolated mitochondria was performed as
described previously (Schwarz et al., 1993), with the
exception of pb-(1-85)-DHFR
(
)(where pb
is precytochrome b
) (Rassow et al., 1990). After the times
indicated, the import reaction was stopped by adding valinomycin to a
final concentration of 2 µM, and unless otherwise
indicated, the samples were subjected to osmotic swelling. The samples
were then split into three aliquots. Aliquot 1 was diluted with 9
volumes of ice-cold SMKCl buffer (250 mM sucrose, 80 mM KCl, 10 mM MOPS/KOH, pH 7.2). Aliquot 2 was diluted with
9 volumes of ice-cold SMKCl buffer containing 100 µg/ml proteinase
K. Aliquot 3 was diluted with 9 volumes of ice-cold hypotonic buffer
(10 mM potassium phosphate, pH 7.5) containing 100 µg/ml
proteinase K. After 30 min on ice, the protease was inactivated by the
addition of phenylmethylsulfonyl fluoride to a final concentration of
1.6 mM and further incubation on ice for 5 min. Mitochondria
from aliquots 1 and 2 and mitoplasts from aliquot 3 were reisolated by
centrifugation. The pellet was solubilized in Laemmli buffer. Protein
was separated by SDS-PAGE (Laemmli, 1970) and blotted onto
nitrocellulose. After autoradiography, data were quantified by laser
densitometry (Ultroscan XL, Pharmacia Biotech Inc.). For the
determination of the matrix-located species, the values obtained were
corrected for the percentage of intact mitochondria still present in
the mitoplast preparation. This was achieved by quantification of the
amount of the protease-protected mature-size form for each time point
with laser densitometry. Chase of the intermediate-size form across the
inner membrane was tested as follows. Import of wild-type precytochrome b
was performed at 12 °C for 10 min. The
mitochondria were reisolated and resuspended in hypotonic swelling
buffer (10 mM potassium phosphate, pH 7.4, 2 µg/ml
trypsin). After 20 min at 0 °C, soybean trypsin inhibitor was added
to a final concentration of 20 times molar excess over trypsin. Then
mitoplasts were further incubated for different time points at 25
°C to allow translocation across the inner membrane. The chase
incubation was performed in the presence of an energy-regenerating
system (final concentrations: 10 µg/ml creatine kinase, 2.5 mM creatine phosphate, 2.5 mM ATP, 2.5 mM NADH, and
5 mM magnesium acetate). For determination of the
mitoplast-located intermediate-size form, the samples were then treated
with 10 µg/ml proteinase K before SDS-PAGE.
Miscellaneous
The recombinant DNA techniques were
as described by Sambrook et al.(1989).
Oligonucleotide-directed mutagenesis, synthesis of radiolabeled
precursor proteins, and immunoblotting were carried out according to
Schwarz et al.(1993). Digitonin fractionation was carried out
by the method of Glick et al. (1992a).
Transient Accumulation of the Intermediate-size Form in
the Matrix Space
To analyze the sorting kinetics at a
submitochondrial level, mitochondria, following the import of
radiolabeled precytochrome b, were subjected to
hypotonic swelling in the presence of exogenously added protease (Glick et al., 1992a; Schwarz et al., 1993). As a control
for efficient opening of the intermembrane space, we used the amount of
the protease-resistant imported mature-size cytochrome b
species in each mitoplast preparation; this serves as an internal
indicator for the remaining intact mitochondria. Thereby, the level of
the matrix-localized intermediate-size species could be specifically
determined.
was
synthesized in reticulocyte lysate and imported into isolated
mitochondria at 25 °C for the times indicated, after which import
was stopped, and the submitochondrial location of the imported species
was determined by hypotonic swelling (Fig. 1, A and B). Import of cytochrome b
and processing
to its mature size occurred in a linear fashion over a time period of
10-20 min, after which it reached a plateau. The
intermediate-size species was observed in intact mitochondria at early
time points; further incubation led to a decrease in this species as it
became processed to the mature-size form. This mature-size species was
exposed to the intermembrane space as it was found to be
protease-sensitive after mitoplast formation. Approximately 25% of the
total imported intermediate-size form was detected as
protease-protected in mitoplasts after 5 min of import (Fig. 1C). The level of this matrix-localized species
declined upon further incubation, which was reflected by a decrease in
the total imported intermediate-size form.
Figure 1:
Import of wild-type
precytochrome b into isolated mitochondria at 25
°C. Import of wild-type precytochrome b
into
isolated mitochondria was performed at 25 °C. At the times
indicated, samples were removed and split into three parts in order to
obtain untreated mitochondria, proteinase K-treated mitochondria, and
mitoplasts as described under ``Materials and Methods.'' A, autoradiograph of the resulting experiment, which was also
quantified by laser densitometry; B, protease-protected
species present in mitochondria; C, amount of
protease-protected species in the matrix after correction for
incomplete swelling. The swelling efficiency was determined by
calculating the amount of the protease-protected mature-size form
present in each of the mitoplast samples. The percentage of the
matrix-located intermediate-size form with respect to the total
imported intermediate-size form is indicated for each time point. M, intact mitochondria; MP, mitoplasts; PK,
proteinase K; p, precursor; i, intermediate; m, mature.
When the import reaction
was performed at 12 °C, the overall import process was slightly
retarded (Fig. 2, A and B). The kinetics of
both formation of and decrease in the intermediate-size form in the
matrix were also slower, with a peak being reached after 15 min. At
this time point, 50% of the total imported intermediate-size
species was localized in the matrix space (Fig. 2C).
Further incubation led to a reduction to about half of its original
level after 90 min of import.
Figure 2:
Import of wild-type precytochrome b at 12 °C. Import was performed as described
for Fig. 1 with the exception that the temperature during import was 12
°C. A, samples subjected to SDS-PAGE and fluorography; B, the protease-protected species in mitochondria; C,
the intermediate-size species in the matrix. The percentage of the
matrix-localized intermediate-size form with respect to the total
imported intermediate-size form is indicated for each time point. The
values have been corrected for incomplete swelling. D, the
intermediate-size species in the matrix space during a second
incubation after reisolation of mitochondria. The intermediate-size
form in the matrix was determined as described for C. M, intact mitochondria; MP, mitoplasts; PK,
proteinase K; p, precursor; i, intermediate; m, mature.
The results demonstrate that a
considerable amount of the total imported intermediate-size form
transiently accumulated in the matrix at early time points. The pool of
the matrix-localized intermediate-size form was increased when import
was performed at a lower temperature. Furthermore, the results show
that the pool of the intermediate-size form protected in the matrix is
not large enough to serve as a source for the total amount of the
mature-size species arising in the intermembrane space. It should also
be pointed out that protease sensitivity of intermediate- and
mature-size forms in mitoplasts does not necessarily indicate that the
complete polypeptide chain is present in the intermembrane space;
rather, it demonstrates that at least a part of the species is exposed
to this location (Gruhler et al., 1995).
Inhibition of the First Processing Results in Transient
Accumulation of Precursor in the Matrix
The addition of metal
chelators such as EDTA and o-phenanthroline inhibits
proteolytic removal of the signal sequence by matrix processing
peptidase and consequently leads to accumulation of uncleaved
precursors (Schmidt et al., 1984; Hartl et al.,
1987). To analyze the effect of processing inhibition on the sorting,
precytochrome b was imported at 12 °C into
isolated mitochondria in the presence of EDTA/o-phenanthroline (Fig. 3, A and B). No intermediate-size form
was observed, suggesting that the mature-size cytochrome b
was derived directly from its precursor form;
alternatively, incomplete inhibition of matrix processing peptidase may
result in a very low amount of the intermediate-size species, that
could be immediately converted into the mature-size form. The kinetics
of formation of the mature-size species was similar to the uninhibited
situation (compare Fig. 2B and 3A). A large
percentage of the imported uncleaved precursor was observed in the
matrix, and the kinetics of its accumulation there and subsequent
decrease were delayed as compared with those of the intermediate-size
form in the uninhibited control (Fig. 3B and
2C). We conclude therefore that processing of the precursor by
matrix processing peptidase is not required for correct sorting of
cytochrome b
.
Figure 3:
Import of wild-type precytochrome b in the presence of metal chelators. Import of
wild-type precytochrome b
into isolated
mitochondria was performed at 12 °C as described for Fig. 1, except
that 1 mMo-phenanthroline and 5 mM EDTA
were present. The levels of protease-protected species within intact
mitochondria (A) and the precursor form in the matrix (B) were determined as described for Fig. 1. The percentage of
the matrix-localized precursor form with respect to the total imported
precursor species is indicated. Determination of the swelling
efficiency and correction for the mitoplast-localized species were as
described for Fig. 1.
Mutations in the Cytochrome b
We investigated
the sorting of several selected mutations in the cytochrome bPresequence
Affect Sorting to the Intermembrane Space
presequence. First, a deletion mutant,
precytochrome b
(
34-46), was studied in
which were eliminated the 13 amino acid residues that precede the basic
amino acid cluster in the sorting signal, which has been demonstrated
to be crucial for the correct sorting of cytochrome b
(Fig. 4) (Beasley et al., 1993; Schwarz et
al., 1993).
Figure 4:
Primary sequence of the cytochrome b targeting and sorting sequence and derived
mutant proteins. The bipartite presequence of cytochrome b
is presented in single letter code. The first
three amino acids of the mature protein are indicated in italics. The two processing sites after the targeting signal
and the sorting sequence are indicated by opened and closedarrowheads, respectively. The point mutation
of Arg to Gly at position 30 (position -2 with respect to the
intermembrane space sorting signal) is indicated by an arrow.
The 13-amino acid deletion in the precytochrome b
-(
34-46) sorting sequence is underlined.
Radiolabeled
pb-(
34-46) was imported into
mitochondria at 25 °C for the times indicated (Fig. 5A). Processing to the mature-size form and
sorting to the intermembrane space were only slightly affected in this
mutant. The mutation resulted in a reduced processing efficiency by
matrix processing peptidase in comparison to the wild-type situation
(see Fig. 1A). Localization experiments revealed that
>30% of the total imported precursor species was present in the
matrix after 5 min of import at 25 °C (Fig. 5B). The
precursor form transiently accumulated in the matrix, and its
appearance and disappearance occurred with kinetics similar to those
observed for the intermediate-size form of wild-type cytochrome b
. The small amount of the intermediate-size
species formed was almost completely maintained in the matrix over a
time period of 45 min, suggesting that it is incompetent for correct
sorting. Thus, the mature-size species is generated directly from the
imported precursor species. The deletion mutant lacks residues required
for the efficient sorting to the intermembrane space. This defect,
however, becomes severe only when processing by matrix processing
peptidase has taken place.
Figure 5:
Import of precytochrome b (
34-46) into isolated mitochondria at 25 °C. A, the protease-protected species within intact mitochondria; B, the matrix-localized precursor and intermediate-size
species. Determination of the levels of the various forms was as
described for Fig. 1. The numbers above the plotted symbols indicate
the percentage of the total imported species.
Mutations close to the matrix processing
peptidase cleavage site of the cytochrome b presequence can result in an inhibition of processing by matrix
processing peptidase. The mutation of Arg to Gly at position 30 in the
matrix targeting sequence of cytochrome b
(two
residues N-terminal to the matrix processing peptidase cleavage site)
has been reported to cause a complete block of matrix processing
peptidase processing in vitro (Arretz et al., 1994).
The sorting kinetics of this mutant precytochrome b
was analyzed at 25 °C. The precursor was imported into
isolated mitochondria, but did not undergo processing to the
mature-size form (Fig. 6A). Rather, it accumulated
unprocessed, with the majority being in the matrix (Fig. 6B). No proteolytic degradation of this
mislocalized species was observed after prolonged incubation periods.
Figure 6:
Import of precytochrome b (Arg-30
Gly) mutant into isolated mitochondria at 25
°C. Import and analysis of the mutant precursor were as described
for Fig. 1. A, the total imported precursor form; B,
the matrix-localized precursor form. Numbers in B indicate the
percentage of the matrix-localized form as related to the total
imported form.
The fact that a mutation N-terminal to the first processing resulted
in missorting to the matrix space can be explained by an altered
conformation of the sorting sequence or by an altered charge
distribution. Thereby, the sorting signal may no longer be recognized
for transport to the intermembrane space.
A Short Fusion Protein of Cytochrome b
The sorting of a fusion protein consisting of the
first 85 amino acids of cytochrome band DHFR Is Partially Imported into the Matrix Space at 15
°C, but Is Correctly Sorted to the Intermembrane Space at 25
°C
and DHFR,
pb
-(1-85)-DHFR, was analyzed. This fusion
protein contains the complete cytochrome b
presequence plus five amino acids of the mature sequence fused to
DHFR. To study the mitochondrial sorting of this fusion protein, we
used a second sublocalization technique, digitonin titration analysis
in the presence of proteinase K, to successively access the
intermembrane space and matrix (Glick et al., 1992a).
-(1-85)-DHFR was imported
into mitochondria at 15 °C, after which the sample was
trypsin-treated and divided. Mitochondria from one-half were
fractionated immediately with digitonin (Fig. 7A, 15
°C), while the other half was further incubated in a chase
reaction at 25 °C and then subjected to digitonin treatment (Fig. 7A, 15 °C
25 °C).
Following import at 15 °C, the fusion protein accumulated largely
as its intermediate-size species. Fractionation of these mitochondria
revealed that a significant proportion of this species (35%) was
present in the matrix. The small amount of the mature-size form was
correctly sorted to the intermembrane space. The matrix-localized
intermediate-size species failed to become chased out of the matrix
into the intermembrane space when exposed to elevated temperatures.
Instead, this intermediate underwent processing to a smaller species,
the i* form (Schwarz et al., 1993). An increase in mature-size b
-(1-85)-DHFR was observed following the
chase reaction. This species is very likely derived from the Imp1p
processing of intermediate b
-(1-85)-DHFR,
which had already been correctly translocated to the intermembrane
space in the first incubation at 15 °C.
Figure 7:
Import of
pb-(1-85)-DHFR at 15 and 25 °C.
Urea-denatured precursor (Stuart et al., 1994) was imported
into 1.5 mg of mitochondrial protein in the presence of 2 mM NADH and 2 mM ATP (final volume of 1.5 ml) either at 15
°C (A) or at 25 °C (B) for 5 min. Following
import, samples were treated with trypsin (25 µg/ml) for 10 min on
ice. After the addition of soybean trypsin inhibitor (125 µg/ml), sampleA was split into two halves, and mitochondria
were reisolated. The mitochondria from one-half of sampleA and those of sampleB were treated
immediately with digitonin in the presence of proteinase K as described
under ``Materials and Methods.'' Mitochondria from the other
half of sampleA were resuspended in import buffer
and further incubated at 25 °C for 20 min in the presence of 2
mM NADH and 2 mM ATP. Mitochondria were then
reisolated and subjected to digitonin treatment in the presence of
proteinase K. Samples were analyzed by SDS-PAGE and were subsequently
blotted onto nitrocellulose and autoradiographed. Blots were
immunodecorated using antibodies against cytochrome b
(Cyt. b
) and cyclophilin (Cpr3p),
endogenous markers for the intermembrane space and matrix,
respectively. C, pb
-(1-85)-DHFR was
imported into mitochondria at either 12 or 25 °C for the time
points indicated. Samples were trypsin-treated, and localization of the
intermediate-size form was performed by hypotonic swelling as described
for Fig. 1. The levels of the intermediate-size species present in the
matrix were determined following densitometry of the resulting
fluorograph and are expressed as a percentage of the total imported
species at each temperature. i, intermediate; m,
mature.
In contrast, when the
same fusion protein was imported into mitochondria at 25 °C,
processing to the intermediate- and mature-size species occurred. Both
forms were located in the intermembrane space (Fig. 7B, 25 °C). These results suggest that correct sorting of this
short fusion protein is temperature-dependent, with a higher proportion
becoming missorted at lower temperatures. This conclusion was supported
by the kinetic analysis of sorting of this protein at the two chosen
temperatures (Fig. 7C). At low temperature (12 °C),
intermediate-size species accumulated in the matrix in a time-dependent
manner. However, at 25 °C, a transient accumulation in the matrix
was observed; this accumulation was much less pronounced than that
observed with, for example, wild-type cytochrome b, thus demonstrating that the levels of
matrix-located intermediates can vary, even between different
preproteins with the same presequence.
-(1-85)-DHFR is not efficiently sorted to
the intermembrane space at 15 °C in contrast to 25 °C. This
result is inconsistent with the stop transfer model of sorting as
import at lower temperatures has been reported to slow down
translocation through the import machinery (Schleyer and Neupert, 1985)
and should therefore enhance the sorting according to a stop transfer
mechanism. Rather, these findings suggest a temperature-sensitive
export process that most likely occurs in parallel with completion of
the import process. If, however, this process becomes temporally
separated by retarding the export step, e.g. at lower
temperatures, it appears that the sorting process cannot resume.
into the
intermembrane space along the conservative sorting pathway has recently
been contested. In an analysis carried out by Glick et al. (1992a), <1% of the imported species was reported to be the
matrix-located intermediate-size form. The authors explained the
discrepancy between their findings and the previously published data
(Hartl et al., 1986, 1987) by the different experimental
techniques. Thus, we have investigated the sorting of cytochrome b
to the intermembrane space by undertaking
detailed kinetic analysis and by applying the mitochondrial
subfractionation procedure developed by Glick et al. (1992a).
presented
above revealed that 25-50% of the imported intermediate-size form
was located in the matrix space at early time points of import.
Concomitant with an increase in the mature-size form in the
intermembrane space, the intermediate-size form in the matrix declined.
The data imply that the decrease in the intermediate-size form in the
matrix is due to a retranslocation into the intermembrane space, with
intermediate-size forms representing sorting intermediates on their way
to the final subcompartment.
has to be initially imported into the matrix space. Upon
emergence in the matrix, the sorting sequence assumes a conformation
that is recognized by a sorting component(s) in the matrix or at the
inner face of the inner membrane. The interaction with this putative
component initiates insertion of the sorting signal into the inner
membrane from the matrix side. Complete import of the intermediate-size
form into the matrix space is not necessarily required for correct
sorting into the intermembrane space. Rather, the prevailing part of
cytochrome b
sorting intermediates probably loops
through the matrix space ``coupling import with export'' and
thus cannot be detected as fully matrix-imported intermediate-size
forms. Accordingly, the 25% of the total imported intermediate-size
forms that transiently accumulated as protease-protected species in the
matrix may represent sorting intermediates that have already engaged in
the retranslocation process. On the other hand, when the
intermediate-size form was accumulated in the matrix at low
temperature, this species could not be chased into the intermembrane
space and was observed to be stably maintained in the matrix. These
data suggest that translocation back across the inner membrane into the
intermembrane space may be coupled with the import process. Once
halted, retranslocation may be unable to resume as folding of segments
of the polypeptide chain in the matrix may prevent further transport.
and thus
should not result in missorting of the precursor into the matrix.
However, as demonstrated, inhibition of matrix processing peptidase by
removal of divalent cations in fact led to import of a substantial
amount of precursor into the matrix space. In contrast to the data
presented by Glick et al. (1992a), but in agreement with our
earlier data (Hartl et al., 1987),
40% of the total
imported precursor was found to be located in the matrix space.
(
34-46), which contains
a deletion of 13 residues preceding the basic amino acid cluster of the
sorting signal. Only the precursor form was able to be translocated
into the intermembrane space; the small amount of the intermediate-size
species formed remained stable in the matrix. Since the decrease in the
matrix-localized mutant precursor form inversely paralleled the
increase in the mature-size form, we conclude that the presence of a
mitochondrial targeting sequence probably suppresses the adverse effect
of the deletion, which renders the intermediate incompetent for
translocation. Again, the stable maintenance of the mutant
intermediate-size form in the matrix indicates that in the case where a
decline in the intermediate-size form was observed for wild-type
preproteins, it reflects a translocation process out of the matrix and
not proteolytic breakdown.
and probably also cytochrome c
would specifically bind to the sorting sequence to ensure their
arrest and thus prevent their complete translocation across the inner
membrane. As demonstrated above, not only mutations in the 50 residues
comprising the sorting signal itself result in precursor accumulation
in the matrix space. Rather, also an exchange at position -2 of
the intermembrane space targeting sequence (two amino acid residues
N-terminal to the matrix processing peptidase cleavage site) led to
mistargeting to the matrix space. This observation can easily be
explained by an alteration in conformation or charge distribution that
no longer allows recognition by the sorting component and/or insertion
into the inner membrane. The fact that the mutation lies N-terminal to
the sorting sequence makes an interpretation according to a stop
transfer pathway difficult.
-(1-85)-DHFR,
resulting in accumulation of missorted species in the matrix under
these conditions. Low temperature should actually enhance arrest in the
translocation channel; and thus, temperature dependence of sorting is
inconsistent with the stop transfer model.
in the matrix in such a
manner that they become inaccessible to protease added to mitoplasts.
This procedure leads to degradation of cytochrome b
sorting intermediates that are completely sorted to the
intermembrane space together with those that are only partially exposed
to this compartment, i.e. that could be still spanning the
inner membrane and undergoing an export event. In a separate study, we
have shown that indeed a large proportion of intermediate- and even
mature-size cytochrome b
species span the inner
membrane and thereby expose segments to the matrix space (Gruhler et al., 1995). These observations are fully consistent with
the conclusion of the present study, namely sorting of cytochrome b
through the mitochondrial matrix.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.