©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sorting of Cytochrome b to the Intermembrane Space of Mitochondria
KINETIC ANALYSIS OF INTERMEDIATES DEMONSTRATES PASSAGE THROUGH THE MATRIX (*)

Hideyu Ono , Albrecht Gruhler , Rosemary A. Stuart , Bernard Guiard (1), Elisabeth Schwarz , Walter Neupert (§)

From the (1)Institut für Physiologische Chemie, Universität München, Goethestrasse 33, D-80336 München, Federal Republic of Germany and the Centre de Génétique Moléculaire CNRS, Université Pierre et Marie Curie, F-91190Gif-sur-Yvette, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Precytochrome b 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.


INTRODUCTION

Cytochromes c 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).

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 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).

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 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.


MATERIALS AND METHODS

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).


RESULTS

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.

Radiolabeled precytochrome b 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).

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.

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 bPresequence Affect Sorting to the Intermembrane Space

We investigated the sorting of several selected mutations in the cytochrome b 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 band DHFR Is Partially Imported into the Matrix Space at 15 °C, but Is Correctly Sorted to the Intermembrane Space at 25 °C

The sorting of a fusion protein consisting of the first 85 amino acids of cytochrome b 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).

Radiolabeled pb-(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.

The data demonstrate that pb-(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.


DISCUSSION

Sorting of cytochrome b 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).

The sorting kinetics of cytochrome b 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.

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 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.

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 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.

Inhibition of precursor processing was also observed for precytochrome b (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.

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 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.

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-(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.

Finally, in this study, we have focused on the transient accumulation of intermediate-size forms of cytochrome b 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.


FOOTNOTES

*
This work was supported by Genzentrum München, Sonderforschungsbereich 184, the Human Frontiers Science Program, and a grant from the Alexander von Humboldt Gesellschaft (to H. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-89-5996-313; Fax: 49-89-5996-270; E-mail: neupert@bio.med.uni-muenchen.de.

The abbreviations used are: DHFR, dihydrofolate reductase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Gabi Ludwig and Sandra Weinzierl for excellent technical assistance.


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