©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitochondrial Import of Subunit Va of Cytochrome c Oxidase Characterized with Yeast Mutants
INDEPENDENCE FROM RECEPTORS, BUT REQUIREMENT FOR MATRIX hsp70 TRANSLOCASE FUNCTION (*)

(Received for publication, October 27, 1994; and in revised form, December 9, 1994)

Frank Gärtner Wolfgang Voos Amparo Querol (1) Brian R. Miller (1)(§) Elizabeth A. Craig (2) Michael G. Cumsky (1) Nikolaus Pfanner (¶)

From the  (1)Biochemisches Institut, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Federal Republic of Germany, the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717, and the (2)Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have investigated the unusual import pathway of cytochrome c oxidase subunit Va (COXVa) into the yeast mitochondrial inner membrane by use of mutants that lack import receptors or are defective in matrix hsp70. (i) Mitochondria lacking the receptor MOM72 are not impaired in import of COXVa. Mitochondria lacking the main receptor MOM19 are moderately reduced in import of COXVa; this, however, is caused by a reduction of the inner membrane potential and not by a lack of specific receptor functions. (ii) Mitochondria defective in the unfoldase function of matrix hsp70 efficiently import COXVa, whereas mitochondria defective in the translocase function of the hsp70 are blocked in import of COXVa. A COXVa construct where the internal hydrophobic sorting signal is placed close to the presequence does not require either hsp70 function.

These results demonstrate that import of COXVa does not require MOM19 or MOM72, but they unexpectedly reveal a strong dependence on the translocase function of matrix hsp70. Two important implications about the characterization of mitochondrial protein import in general are obtained. First, the interpretation of import results with mutants lacking MOM19 have to consider effects on the membrane potential. Second, the distance between a matrix targeting sequence and a hydrophobic sorting sequence within a precursor appears to determine if the inner membrane sorting machinery can substitute for the translocase function of hsp70 or not.


INTRODUCTION

The majority of nuclear-encoded mitochondrial preproteins are synthesized with amino-terminal presequences that are characterized by a prevalence of positively charged amino acid residues and the potential to form an amphipathic alpha-helix. The presequences direct translocation of the polypeptide chains into or across the mitochondrial inner membrane (``matrix targeting sequence'') and are cleaved off by the processing peptidase in the matrix (Douglas et al., 1986; Hartl et al., 1989; Horwich, 1990; Baker and Schatz, 1991; Pfanner et al., 1994). Nearly all these cleavable preproteins analyzed so far require receptor proteins on the outer membrane surface and the heat shock protein hsp70 in the matrix (mt-hsp70) (^1)for import into mitochondria.

Initial characterization of the biogenesis of subunit Va of cytochrome c oxidase (COXVa) suggested an exceptional behavior. Import of COXVa (i) was not inhibited by pretreatment of mitochondria with a protease known to degrade the surface receptors (Miller and Cumsky, 1991) and (ii) was not inhibited by a mutation in mt-hsp70 (Miller and Cumsky, 1993). COXVa appears to follow a novel targeting and sorting pathway into the mitochondrial inner membrane. However, alternative possibilities to explain the surprising results obtained with the import of COXVa are as follows. (i) The treatment of isolated mitochondria with protease not only removes the surface receptors MOM19 (MAS20) and MOM72 (MAS70), but may open cryptic import sites that allow non-physiological import of certain preproteins. Therefore, an import inhibition by removal of the surface receptors may be partially or completely reversed. To exclude this possibility, a selective removal of surface receptors is needed. (ii) The import of COXVa was not affected by a mutation in mt-hsp70 that inhibits import of other mitochondrial preproteins (Kang et al., 1990; Miller and Cumsky, 1993). A recent analysis assigned two distinct functions to mt-hsp70 in import of preproteins, facilitation of unfolding of preproteins during translocation (unfoldase function), and actual translocation across the inner membrane independently of the folding state of preproteins (translocase function) (Gambill et al., 1993; Voos et al., 1993). As the mutant mitochondria used were found to be solely defective in the unfoldase function (Gambill et al., 1993), it cannot be excluded that import of COXVa depends on the translocase function of mt-hsp70.

Here we tried to answer these questions about the import pathway of COXVa and to set its import into context with the general mitochondrial import pathway. To circumvent possible limitations of a biochemical analysis of the role of import receptors, we studied the biogenesis of COXVa with Saccharomyces cerevisiae mutants selectively lacking either MOM19 or MOM72 (Moczko et al., 1994). Moreover, we analyzed COXVa import in a temperature-sensitive S. cerevisiae strain defective in the translocase function of mt-hsp70 (Gambill et al., 1993). We show that import of COXVa (i) is indeed independent of the receptor functions of MOM19 or MOM72, but (ii) strictly requires the translocase function of mt-hsp70. A comparison of COXVa import with the general mitochondrial import pathway leads to important implications on the characterization of receptor mutants and the mechanisms of inner membrane protein sorting.


MATERIALS AND METHODS

S. cerevisiae Strains

Construction of the receptor mutants DeltaMOM19 and DeltaMOM72 by gene disruption as well as of the temperature-sensitive mt-hsp70 mutants ssc1-2 and ssc1-3 has been described previously (Moczko et al., 1994; Gambill et al., 1993). The haploid yeast strains of the mutants and their corresponding wild types are indicated in Table 1.



Protein Import into Isolated Yeast Mitochondria

The S. cerevisiae strains were grown in YP medium (1% Bacto-yeast extract, 2% Bacto-peptone) containing 2% glucose (receptor mutants) or 3% glycerol (mt-hsp70 mutants), and mitochondria were isolated according to published procedures (Daum et al., 1982; Hartl et al., 1987; Kang et al., 1990; Gambill et al., 1993). Mitochondrial precursor proteins were synthesized in rabbit reticulocyte lysates in the presence of [S]methionine as described (Söllner et al., 1991). COXVa constructs lacked the amino acid residues 101-118 (COXVa; Glaser et al., 1990) or 26-89 (COXVa) (^2)of the COXVa precursor. The standard import assays included isolated yeast mitochondria (50 µg of protein), radiolabeled precursor (5 µl of reticulocyte lysate), 2 mM Mg-ATP, and 2 mM NADH in bovine serum albumin (BSA)-containing buffer (Söllner et al., 1991) at a final volume of 0.1 ml. The reactions were incubated for 3-5 min at 25 °C (within the kinetically linear range) and stopped with 1 µM valinomycin. Dissipation of the membrane potential Delta was achieved by adding 1 µM valinomycin and 20 µM oligomycin prior to incubation. Published procedures (Söllner et al., 1991) were used for proteinase K treatment, reisolation of the mitochondria, separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), densitometric analysis of fluorograms, or storage phosphorimaging analysis (Molecular Dynamics, Inc.) of autoradiograms.

Assessment of the Mitochondrial Inner Membrane Potential Delta

The membrane potential Delta of isolated yeast mitochondria was assessed using the potential-sensitive fluorescent dye 3,3`-dipropylthiadicarbocyanine iodide (DiSC(3)(5); Molecular Probes, Inc.). The method is based on the potential-dependent partitioning of the dye between mitochondria and the medium, leading to a decrease in fluorescence (``quenching'') with increasing Delta (Sims et al., 1974). The measurements were performed with a Perkin-Elmer LS 50B luminescence spectrometer at 25 °C, excitation at 622 nm, emission at 670 nm, slits 5 nm. Successively, the following reagents were added to a cuvette and the fluorescence changes registered in arbitrary units: 3 ml of buffer (0.6 M sorbitol, 0.1% BSA, 10 mM MgCl(2), 0.5 mM EDTA, 20 mM KP(i), pH 7.4), 3 µl of DiSC(3)(5) (in Me(2)SO; 2 µM final concentration), 20 µl of mitochondria in SEM buffer (250 mM saccharose, 1 mM EDTA, 10 mM MOPS, pH 7.2; 33 µg/ml protein final concentration), and finally 3 µl of KCN (1 mM final concentration) + 3 µl of valinomycin (in ethanol; 1 µM final concentration) to dissipate Delta. The difference between the fluorescence prior to and after addition of KCN/valinomycin represents a relative assessment of the mitochondrial membrane potential. The isolated mitochondria of wild-type and mutant cells apparently contained sufficient internal substrates as the inclusion of external substrates (2 mM NADH plus 2 mM ATP, or 5 mM succinate plus 5 mM malate) did not lead to further increase of fluorescence quenching.

CCCP Titration

For gradually reducing the membrane potential by stepwise uncoupling, increasing concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma; from a 100-fold concentrated stock solution in ethanol) were included in the import reactions (Martin et al., 1991). In order to prevent a decrease of ATP levels and generation of an electrochemical potential by a reversed action of the F(0)F(1)-ATPase, 20 µM oligomycin was present in the assay.


RESULTS

Import of COXVa Is Not Affected in DeltaMOM72 Mitochondria but Is Moderately Reduced in DeltaMOM19 Mitochondria

The precursor of COXVa was synthesized in rabbit reticulocyte lysates in the presence of [S]methionine and incubated with energized isolated S. cerevisiae mitochondria in the kinetically linear import range. To assess the import of COXVa, the mitochondria were then treated with protease, reisolated, and analyzed by SDS-PAGE.

Import of COXVa into mitochondria from a strain lacking MOM72 was analyzed. The import occurred with comparable efficiency into DeltaMOM72 and wild-type mitochondria (Fig. 1A, columns1 and 2). As controls for the specific effect of the deletion of MOM72, we show that import of the non-cleavable preprotein ADP/ATP carrier is inhibited (Fig. 1A, column4), while the import of cleavable preproteins is slightly or not inhibited (Moczko et al., 1993, 1994). Cleavable preproteins used were the alpha-subunit of the mitochondrial processing peptidase (alpha-MPP) and a fusion protein between the presequence of F(0)-ATPase subunit 9 and dihydrofolate reductase (Su9-DHFR) (Fig. 1A, columns6 and 8).


Figure 1: Import of COXVa into mutant mitochondria lacking MOM72 or MOM19. A, COXVa import into DeltaMOM72 mitochondria occurs with wild-type efficiency. Isolated, energized (2 mM ATP, 2 mM NADH) mitochondria from wild-type (WT) or DeltaMOM72 yeast (50 µg of protein) were incubated for 5 min at 25 °C with rabbit reticulocyte lysates containing the radiolabeled precursor proteins of COXVa, the ADP/ATP carrier (AAC), alpha-MPP, or Su9-DHFR. Import was stopped with 1 µM valinomycin, and after protease treatment (200 µg/ml proteinase K) of the mitochondria, reisolation, and separation on SDS-polyacrylamide gels, fluorograms were analyzed by densitometry. The import into wild-type mitochondria was set to 100%, respectively. B, a moderate import reduction of COXVa in DeltaMOM19 mitochondria. The experiment was performed with the precursors of COXVa, alpha-MPP, and Su9-DHFR as described above, except that DeltaMOM19 mitochondria were employed and a protease treatment was omitted. A similar relation of import into wild-type and DeltaMOM19 mitochondria was observed, when the mitochondria were treated with proteinase K after the import reaction. p, precursor; i, intermediate; m, mature form of a protein.



The import of COXVa into DeltaMOM19 mitochondria, however, was moderately reduced to about two-thirds of the import efficiency into wild-type mitochondria (Fig. 1B, lanes1 and 2). The reduction was significant as determined from the standard error of the means of several independent experiments (Table 2). The import of preproteins such as alpha-MPP and Su9-DHFR, which are known to be imported via MOM19 (Moczko et al., 1993), was more strongly inhibited by a deletion of MOM19 (Fig. 1B, lanes 3-6; Table 2).



The Reduction of COXVa Import into DeltaMOM19 Mitochondria Is Due to a Reduction of the Membrane Potential

The moderate reduction of COXVa import into DeltaMOM19 mitochondria could be due to a direct requirement of import for MOM19 or could be caused by an indirect effect of the MOM19 deletion on other steps of the COXVa import pathway. Import of COXVa was shown to require a membrane potential Delta across the mitochondrial inner membrane (Glaser et al., 1990). By use of the fluorescent dye 3,3`-dipropylthiadicarbocyanine iodide (DiSC(3)(5)), we assessed if DeltaMOM19 mitochondria possessed a membrane potential Delta. The decrease in fluorescence after addition of mitochondria shows that DeltaMOM19 mitochondria were able to generate a Delta (Fig. 2). The fluorescence quenching of DeltaMOM19 mitochondria, however, was less than that of wild-type mitochondria, indicating that DeltaMOM19 mitochondria possessed a lower membrane potential than wild-type mitochondria (Fig. 2; Table 2). The Delta reduction was fully reversible when MOM19 was expressed from a plasmid in the DeltaMOM19 strain (not shown).


Figure 2: Assessment of the membrane potential in mitochondria lacking MOM19. The membrane potential Delta of isolated DeltaMOM19 and wild-type mitochondria was determined by fluorescence photometry with the potential-sensitive dye DiSC(3)(5) as described under ``Materials and Methods.'' A pronounced fluorescence quenching (decrease) after addition of the mitochondria (100 µg of protein) compared to the levels obtained with 1 µM valinomycin/1 mM KCN (in order to dissipate the membrane potential) indicates a high Delta.



Different preproteins can have different requirements for the magnitude of the membrane potential needed to drive import (Martin et al., 1991). It can be argued that import of COXVa has the same dependence on MOM19 as import of alpha-MPP and Su9-DHFR and the weaker inhibition of its import into DeltaMOM19 mitochondria is just due to a lower Delta-requirement. To exclude this, we asked how the Delta-dependence of COXVa import was related to that of alpha-MPP and Su9-DHFR. Wild-type mitochondria were partially uncoupled by limiting concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). The higher the CCCP concentration needed for a half-maximal inhibition of import, the lower is the membrane potential needed for import (Martin et al., 1991). COXVa import showed a CCCP inhibition curve similar to that of alpha-MPP, whereas Su9-DHFR could still be imported at lower membrane potentials (Fig. 3). Therefore, the Delta-dependence of COXVa is comparable to that of alpha-MPP and even higher than that of Su9-DHFR. When the import efficiencies of the three preproteins into DeltaMOM19 mitochondria were corrected for the reduction of fluorescence quenching, i.e. the relative assessment of the membrane potential, the import of COXVa was of wild-type efficiency (Table 2). As expected, the import of alpha-MPP and Su9-DHFR was still strongly inhibited, in agreement with a dependence of their import on the receptor function of MOM19 (Table 2).


Figure 3: Dependence of COXVa import on the mitochondrial membrane potential. Isolated wild-type mitochondria (50 µg of protein) were suspended in 1% BSA-containing import buffer in the presence of 2 mM ATP, 2 mM NADH, and 20 µM oligomycin and partially uncoupled by adding increasing concentrations of the protonophore CCCP as indicated (see ``Materials and Methods''). Then the S-labeled precursor proteins COXVa, alpha-MPP, and Su9-DHFR were imported for 5 min at 25 °C, and the reactions were stopped with 1 µM valinomycin. After reisolation and SDS-PAGE separation of the mitochondria, the processed proteins were quantified by densitometry of the fluorograms. Samples receiving no CCCP represented 100% import efficiency.



We conclude that the reduction of COXVa import into DeltaMOM19 mitochondria is indirectly caused by a reduction of the membrane potential and that the import of COXVa does not require the receptors MOM72 and MOM19. In support of this, antibodies directed against MOM19 or MOM72 (Moczko et al., 1993) did not inhibit import of COXVa into isolated mitochondria (not shown).

Import of COXVa Depends on the Translocase Function of mt-hsp70

Two temperature-sensitive mutant strains of mt-hsp70 (ssc1-2 and ssc1-3) were used. The phenotypes are induced by shifting the isolated mitochondria to 37 °C (Gambill et al., 1993). ssc1-2 mitochondria are defective only in the unfoldase function of mt-hsp70, that is the mutant hsp70 (Ssc1-2p) does not promote unfolding of polypeptide chains during membrane translocation, yet has sufficient binding capacity to drive translocation of an unfolded preprotein into the matrix. Ssc1-3p is unable to bind to precursor polypeptides and therefore is defective in both the unfoldase function and the translocase function (Voos et al., 1993).

In agreement with a previous report (Miller and Cumsky, 1993), import of COXVa into ssc1-2 mitochondria was not inhibited (Fig. 4, lanes4 and 10). However, import into ssc1-3 mitochondria was blocked (Fig. 4, lanes6 and 12). Proteolytic processing of the presequence was also inhibited with ssc1-3 mitochondria (Fig. 4, lane6), indicating that binding to mt-hsp70 is needed to drive COXVa deep enough into mitochondria to expose the cleavage site to the matrix processing peptidase. To exclude unspecific inhibitory effects of the ssc1-3 mitochondria on Delta-dependent translocation or on processing of preproteins, we analyzed the import of a special test protein, a fusion between the 167 amino-terminal amino acid residues of the precursor of cytochrome b(2) and entire dihydrofolate reductase (b(2)-DHFR). This fusion protein has been shown to be imported independently of functional mt-hsp70. The presequence of cytochrome b(2) is bipartite. A typical matrix targeting sequence in the first half of the presequence is followed by a second sorting sequence which includes a hydrophobic core and is required for directing transport of cytochrome b(2) to the intermembrane space. A hypothetical sorting component is assumed to bind to the sorting sequence and to substitute for the translocase function of mt-hsp70 (Voos et al., 1993; Glick et al., 1993; Stuart et al., 1994). Unfolding of b(2)-DHFR in the in vitro system seems to need only low energy inputs, not requiring the unfoldase function of mt-hsp70 (Voos et al., 1993). The import of b(2)-DHFR into ssc1-2 and ssc1-3 mitochondrial preparations used in this study was not affected (Fig. 4), demonstrating the import capabilities of the mutant mitochondria as long as functional mt-hsp70 is not required. We conclude therefore that import of COXVa requires the translocase function of mt-hsp70.


Figure 4: Import of COXVa into ssc1-3 mitochondria is strongly inhibited. Mitochondria from wild-type yeast and from the temperature-sensitive mt-hsp70 mutants ssc1-2 and ssc1-3 (50 µg of protein) were shifted to 37 °C for 10 min in order to stably induce their phenotypes (Gambill et al., 1993). 1 µM valinomycin and 20 µM oligomycin dissipated the membrane potential where indicated. Then the import of radiolabeled COXVa precursor protein and of a fusion protein between the 167 amino-terminal residues of the cytochrome b(2) precursor and entire dihydrofolate reductase (b(2)-DHFR) into the energized mitochondria was performed for 3 min at 25 °C and stopped by addition of 1 µM valinomycin. One half of each sample was treated with 100 µg/ml proteinase K. The mitochondria were reisolated, subjected to SDS-PAGE, and analyzed by fluorography and densitometry. The amount of mature-sized protein in wild-type mitochondria was set to 100%. The faint protease-sensitive band below mature COXVa (and COXVa in Fig. 5) is already present in the reticulocyte lysate and is probably due to internal initiation of translation (Hartl et al., 1987). p, precursor; m, mature protein; Delta, membrane potential.




Figure 5: COXVa with a deletion of the internal sorting signal is inhibited in import into ssc1-3 mitochondria. The experiment was performed essentially as described in the legend of Fig. 4with the exception that a COXVa construct carrying a deletion of amino acid residues 101-118 was employed. p, precursor; m, mature protein; Delta, membrane potential.



Requirement for the Translocase Function of mt-hsp70 Is Determined by the Position of the Sorting Sequence within COXVa

To analyze which properties of the precursor protein determine the dependence of COXVa import on the translocase function of mt-hsp70, we tested the import of several COXVa constructs into ssc1 mutant mitochondria.

COXVa contains a hydrophobic segment in its carboxyl-terminal portion (residues 98-119) that is required for directing the polypeptide into the mitochondrial inner membrane (Glaser et al., 1990). A COXVa construct lacking most of this inner membrane sorting sequence (COXVa) was imported into the matrix (Glaser et al., 1990). COXVa showed the same dependence on the functions of mt-hsp70 as wild-type COXVa, i.e. import of COXVa into ssc1-2 mitochondria was not significantly affected (Fig. 5, lanes4 and 10), while import into ssc1-3 mitochondria was inhibited (Fig. 5, lanes6 and 12). Therefore, deletion of the sorting sequence does not influence the dependence of COXVa import on the translocase function of mt-hsp70.

We then employed a construct where a major portion of the sequence between the COXVa presequence (first 20 residues) and the hydrophobic sorting signal was removed. In this construct (COXVa) the sorting signal was placed close to the presequence such that the matrix targeting signal and the sorting signal were contained within the first 60 amino acid residues of the preprotein. The close vicinity of the two distinct signals resembles the situation of bipartite presequences of, for example, cytochrome b(2). The construct COXVa was imported into isolated wild-type yeast mitochondria in a Delta-dependent manner (Fig. 6A, lanes 1 and 2). To test if COXVa was correctly inserted into the mitochondrial inner membrane, we employed a specific assay developed for authentic COXVa (Miller and Cumsky, 1991, 1993). After opening of the intermembrane space by swelling of the mitochondria and addition of proteinase K, only correctly localized COXVa gives rise to a fragment that is about 3 kDa smaller than COXVa. The fragmentation indicates removal of the carboxyl-terminal tail of COXVa behind the hydrophobic sorting and membrane anchor sequence (Miller and Cumsky, 1991, 1993). Fig. 6B (lane2) shows that this typical fragment was also formed from imported COXVa. Probably due to the modification of COXVa close to the cleavage site of the matrix processing peptidase, removal of the amino-terminal presequence from the construct was slow and was thus not observed in the short import times used here (the short import times are necessary to be in the kinetically linear range for membrane translocation). At longer incubation times in the import reaction, the expected proteolytic processing took place (not shown).


Figure 6: Placement of the sorting signal close to the presequence renders import of COXVa independent of functional mt-hsp70. A, import of COXVa into the ssc1-mutant mitochondria. A COXVa construct lacking amino acid residues 26-89 was imported into wild-type, ssc1-2 and ssc1-3 mitochondria as described in the legend of Fig. 4. All samples were treated with 100 µg/ml proteinase K. Imported COXVa (precursor form) is indicated by an arrow. Delta, membrane potential. B, characteristic fragmentation of correctly imported COXVa. The COXVa construct was imported as mentioned above. After reisolating, the mitochondria in one half of each sample were converted to mitoplasts by swelling in 25 mM saccharose, 1 mM EDTA, 10 mM MOPS, pH 7.2 (Blom et al., 1993), reisolated again, and incubated with 100 µg/ml proteinase K in BSA-containing import buffer. The mitochondria were collected by centrifugation and separated by SDS-PAGE. The proteolytic fragment formed by proteinase K is indicated by an asterisk.



COXVa was also efficiently imported to a protease-protected location in ssc1-2 and ssc1-3 mitochondria (Fig. 6A, lanes4 and 6). The import was inhibited by dissipation of the membrane potential Delta (Fig. 6A, lanes3 and 5). The insertion of COXVa into the mitochondrial inner membrane, assessed by formation of the proteolytic fragment, was indistinguishable among wild-type, ssc1-2, and ssc1-3 mitochondria (Fig. 6B, lanes2, 4, and 6). The import behavior of COXVa into ssc1-3 mitochondria is thus in strict contrast to that of authentic COXVa and COXVa.


DISCUSSION

S. cerevisiae mutants represent a powerful system to study intracellular protein sorting. Here we have characterized the unusual mitochondrial import pathway of cytochrome c oxidase subunit Va with yeast mutants defective in distinct import components. Our findings suggest a refined model of COXVa import and provide new insights into the properties of import receptor mutants and the mechanisms of membrane translocation driven by matrix hsp70.

Independence of COXVa Import from the Import Receptors MOM19 and MOM72

Import of COXVa into DeltaMOM72 mitochondria was of the same efficiency as that into wild-type mitochondria, demonstrating that MOM72 is not required for import of this preprotein. However, the import of COXVa was moderately yet significantly reduced with DeltaMOM19 mitochondria. This finding was unexpected, inasmuch as a removal of surface receptors by protease did not reduce COXVa import (Miller and Cumsky, 1991), raising the possibility of opening cryptic import sites by the protease treatment. Alternatively, a deletion of MOM19 may affect further import steps, which are not directly linked to receptor function but which require components imported via MOM19. Evidence for the second possibility was provided by the observation that the membrane potential across the inner membrane of DeltaMOM19 mitochondria was moderately reduced. The reduced import of COXVa correlated with the reduced membrane potential, whereas the import of other presequence-carrying preproteins into DeltaMOM19 mitochondria was more strongly inhibited. After correction of the import efficiencies for the reduction of Delta, import of COXVa was of wild-type efficiency, whereas import of the other cleavable preproteins was of the level observed for an inactivation of surface receptors by other means (inhibitory antibodies and proteases) (Söllner et al., 1989; Moczko et al., 1993). While each of the three approaches to inactivate mitochondrial surface receptors (protease treatment, inhibitory antibodies, and deletion mutants) has its potential pitfalls, the full agreement of the results obtained with the different approaches provides convincing evidence. We conclude that import of COXVa does not require the two known surface receptors MOM19 and MOM72.

Now three mitochondrial preproteins are known that depend upon neither MOM19 nor MOM72 for import. (i) Apocytochrome c seems to be translocated through the lipids of the outer membrane and is trapped in the intermembrane space by covalent attachment of heme mediated by cytochrome c heme lyase (Nicholson et al., 1988; Dumont et al., 1991; Jordi et al., 1992). (ii) The precursor of MOM19 is imported into the receptor complex of the outer membrane without a need for mature MOM19 or MOM72, but directly and stably assembles with MOM38 (ISP42), the major component of the general insertion pore in the receptor complex (Schneider et al., 1991). (iii) COXVa seems to use yet another mechanism for translocation across the outer membrane. To date, COXVa is the only presequence-carrying preprotein that requires neither MOM19 nor MOM72. The presequence of COXVa is positively charged, but is not predicted to form an amphipathic alpha-helix found with typical mitochondrial presequences, which have positive charges clustered on one side of a helix and hydrophobic residues clustered on the other side (Roise et al., 1986; von Heijne, 1986). Recent studies reinforced the importance of an amphipathic alpha-helix for targeting of preproteins via MOM19 (Brink et al., 1994). Other positively charged sequences were shown to mediate a receptor-independent and low efficient ``bypass'' import into mitochondria, which seems to be mediated by the general insertion pore of the receptor complex (Baker and Schatz, 1987; Pfaller et al., 1989). Typical mitochondrial preproteins are also able to use this bypass for a small fraction of their import in addition to the much more efficient receptor-mediated import (Pfaller et al., 1989). The efficiency of COXVa import is far above that observed for bypass import. We speculate that the precursor of COXVa possesses a special ability for direct insertion into and translocation through the general insertion pore of the outer membrane and thus is imported without a need for surface receptors.

Implications for Characterization of Import Receptor Mutants

Mitochondria depleted of MOM19 are currently used widely to characterize the functions of mitochondrial import receptors (Ramage et al., 1993; Harkness et al., 1994; Lithgow et al., 1994; Moczko et al., 1994). The finding reported here has important implications for the interpretation of these results, as it demonstrates a reduction of the membrane potential in DeltaMOM19 mitochondria. The reduction varies between different batches of mitochondria and mutant strains, but in each case correlates with the reduction of COXVa import. (^3)We conclude that the membrane potential of DeltaMOM19 mitochondria must be assessed before interpretation of import inhibition results. A comparison of the results obtained with the three different approaches of inactivation of surface receptors indicates that 70-85% of import of a typical presequence-carrying preprotein occurs via MOM19 (Pfaller et al., 1989; Söllner et al., 1989; Steger et al., 1990; Moczko et al., 1993, 1994; this study). Stronger import inhibitions into DeltaMOM19 mitochondria (Harkness et al., 1994; Lithgow et al., 1994) may be caused by a reduction of Delta. Harkness et al.(1994) reported that their mitochondria depleted of MOM19 were deficient in cristae membranes and cytochromes, suggesting that inner membrane functions such as generation of Delta may be affected. Studies with the control protein mostly used so far, the ADP/ATP carrier, can be misleading as this preprotein can be imported at considerably lower membrane potentials than most cleavable preproteins (Pfanner and Neupert, 1985; Martin et al., 1991). We recommend to use the import of COXVa as internal standard for the quality of DeltaMOM19 mitochondria, as we found that COXVa has a Delta-dependence comparable to that of typical presequence-carrying preproteins, but not the dependence on MOM19.

Dependence of COXVa Import on Matrix hsp70

While previous work suggested that COXVa import was independent of the molecular chaperones hsp70 and hsp60 in the matrix, we found a strong dependence of import on the translocase function of mt-hsp70. In the original temperature-sensitive mutant of mt-hsp70, the chaperone exhibits a residual binding activity for preproteins and thus is able to import loosely folded preproteins (Kang et al., 1990; Gambill et al., 1993; Voos et al., 1993). The precursor of COXVa synthesized in vitro seems to be loosely folded (Miller and Cumsky, 1991, 1993) and does not require the unfoldase function of mt-hsp70. In another temperature-sensitive mutant (ssc1-3), mt-hsp70 is unable to bind to precursor polypeptides, and therefore translocation of preproteins across the inner membrane is blocked even if they are unfolded (Gambill et al., 1993). COXVa was strongly inhibited in import into these mutant mitochondria, indicating that it requires the translocase function of mt-hsp70. Removal of the presequence of COXVa was blocked in the ssc1-3 mitochondria, demonstrating that the presequence is not transported deep enough into the matrix to allow access of the processing peptidase (the activity of the processing peptidase is not affected by the ssc1-3 mutation (Gambill et al., 1993; Voos et al., 1993)). Moreover, no accumulation of uncleaved protease-protected COXVa was observed in the mutant mitochondria, even after prolonged incubation times,^3 indicating a requirement for the translocase function of mt-hsp70 also at the outer membrane level. Therefore, the translocase function of mt-hsp70 appears to be essential for both transfer of COXVa across the outer membrane and into the inner membrane.

Requirement for the Translocase Function of mt-hsp70 Is Influenced by the Position of the Hydrophobic Sorting Sequence

COXVa contains a 20-residue presequence (matrix targeting sequence) and a hydrophobic sequence in the carboxyl-terminal half, which functions as an inner membrane sorting and membrane anchor sequence (Glaser et al., 1990). Some preproteins targeted to the intermembrane space or the outer side of the inner membrane such as cytochrome b(2) and cytochrome c(1) also contain a matrix targeting sequence and a sorting sequence with a hydrophobic core. In these preproteins both signal sequences are located within a bipartite presequence. The import of cytochrome b(2), and possibly cytochrome c(1), was found to be independent of the translocase function of mt-hsp70 (Voos et al., 1993; Glick et al., 1992). In case of cytochrome b(2), the sorting sequence in the second half of the presequence is responsible for independence from the translocase function (Voos et al., 1993). It is assumed that an unidentified component of the inner membrane sorting machinery specifically binds to the sorting sequence and supports translocation of the precursor polypeptide. Thus, the sorting machinery substitutes for the translocase function of mt-hsp70 and allows processing and full import of fusion proteins containing the presequence of cytochrome b(2) into ssc1-3 mitochondria (Voos et al., 1993). We wondered if the sorting sequence of COXVa may play roles in import similar to that of the sorting sequences in the bipartite presequences. Therefore, we moved the sorting sequence of COXVa closer to the presequence to mimic the spatial situation found in bipartite presequences (we previously showed that the length of a preprotein does not influence the dependence on the translocase function (Voos et al., 1993)). This construct was efficiently imported by mitochondria defective in the translocase function of mt-hsp70 and was correctly inserted into the inner membrane, demonstrating that the dependence of COXVa import on mt-hsp70 can be completely changed by changing the intramolecular position of the hydrophobic sorting sequence.

We propose the following model for the inner membrane translocation of preproteins containing a matrix targeting sequence and a hydrophobic sorting sequence (Fig. 7A). The polypeptides are translocated as linear chains. The membrane potential Delta is required to drive translocation of the amino-terminal presequence across the inner membrane but is usually not sufficient to transport the presequence deep enough into the matrix to allow proteolytic cleavage. mt-hsp70 binds to the preprotein segments emerging on the matrix side and thereby promotes translocation of the complete presequence and of mature portions of the preprotein. A component of the inner membrane sorting machinery (MSM) selectively binds to the sorting sequence to direct it into the inner membrane. Binding of the sorting component can fully substitute for the translocase function of mt-hsp70 when the sorting signal is close to the presequence (Fig. 7C). That is, the initial Delta-driven translocation must bring the sorting sequence close enough to the inner membrane sorting machinery such that it can be recognized and bound. It is unknown what drives the proposed translocase function of the sorting machinery, which is able to function at very low ATP levels (Glick et al., 1993; Stuart et al., 1994). Possibly, the energy for driving import is derived from binding of the sorting sequence to the sorting machinery and its subsequent stable insertion into the lipid phase. In case the sorting sequence is too far away from the presequence, the Delta-driven import of the linear polypeptide chain is not sufficient to promote movement of the sorting signal toward the sorting machinery (Fig. 7B), and therefore mt-hsp70 with its broad reactivity for unfolded segments is essential to drive translocation by binding to preprotein segments located closer to the presequence.


Figure 7: Hypothetical model about the role of matrix hsp70 and the inner membrane sorting machinery in import of preproteins with dual targeting information. A, the model shows import of COXVa into the mitochondrial inner membrane driven by a stepwise action of the membrane potential Delta on the presequence (positively charged box), binding of mt-hsp70 molecules to unfolded preprotein segments, and binding of the mitochondrial inner membrane sorting machinery to the hydrophobic sorting sequence (hatchedbox). B, when a mutated mt-hsp70 is unable to bind to preproteins, MSM is not able to bind to the preprotein as the distance to the sorting sequence is too large. Translocation is blocked. C, when the sorting sequence is moved close to the presequence, the Delta-driven translocation transports the sorting sequence close enough to MSM such that binding is possible also in the absence of functional mt-hsp70. Binding to MSM can now substitute for the translocase function of mt-hsp70. GIP, general insertion pore in the outer membrane; IM, inner membrane; IMS, intermembrane space; MIM, mitochondrial inner membrane import machinery; MPP, mitochondrial processing peptidase; OM, outer membrane. The exact topology of the mitochondrial sorting machinery is unknown. The simplified model shown here assumes a location of MSM for COXVa in the inner membrane. A substitution of MSM for the translocase function of mt-hsp70 may also be possible when MSM is located on the matrix side of the inner membrane.




FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. 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.

§
Present address: Dept. of Biology, University of California San Diego, La Jolla, California 92093

To whom correspondence should be addressed. Tel.: 49-761-203-5224; Fax: 49-761-203-5261.

(^1)
The abbreviations used are: mt-hsp70, mitochondrial matrix heat shock protein of 70 kDa (also termed Ssc1p); COXVa, cytochrome c oxidase subunit Va; Delta, membrane potential; MOM19, MOM72, mitochondrial outer membrane proteins (import receptors) of about 19 and 72 kDa, respectively (also termed MAS20 and MAS70, respectively); MPP, mitochondrial processing peptidase; Su9-DHFR, fusion protein between the presequence of F(0)-ATPase subunit 9 and dihydrofolate reductase; MSM, membrane sorting machinery; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DiSC(3)(5), 3,3`-dipropylthiadicarbocyanine iodide; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
A. Querol, B. R. Miller and M. G. Cumsky, unpublished data.

(^3)
F. Gärtner and N. Pfanner, unpublished.


ACKNOWLEDGEMENTS

We are grateful to L. Jung, M. Moczko, and V. Zara for experimental advice and for supplying plasmids and yeast strains.


REFERENCES

  1. Baker, A., and Schatz, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3117-3121 [Abstract]
  2. Baker, K. P., and Schatz, G. (1991) Nature 349, 205-208 [CrossRef][Medline] [Order article via Infotrieve]
  3. Blom, J., Kübrich, M., Rassow, J., Voos, W., Dekker, P. J. T., Maarse, A. C., Meijer, M., and Pfanner, N. (1993) Mol. Cell. Biol. 13, 7364-7371 [Abstract]
  4. Brink, S., Flügge, U.-I., Chaumont, F., Boutry, M., Emmermann, M., Schmitz, U., Becker, K., and Pfanner, N. (1994) J. Biol. Chem. 269, 16478-16485 [Abstract/Free Full Text]
  5. Daum, G., Böhni, P. C., and Schatz, G. (1982) J. Biol. Chem. 257, 13028-13033 [Abstract/Free Full Text]
  6. Douglas, M. G., McCammon, M., and Vassarotti, A. (1986) Microbiol. Rev. 50, 166-178
  7. Dumont, M. E., Cardillo, T. S., Hayes, M. K., and Sherman, F. (1991) Mol. Cell. Biol. 11, 5487-5496 [Medline] [Order article via Infotrieve]
  8. Gambill, B. D., Voos, W., Kang, P. J., Miao, B., Langer, T., Craig, E. A., and Pfanner, N. (1993) J. Cell Biol. 123, 109-117 [Abstract]
  9. Glaser, S. M., Miller, B. R., and Cumsky, M. G. (1990) Mol. Cell. Biol. 10, 1873-1881 [Medline] [Order article via Infotrieve]
  10. Glick, B. S., Brandt, A., Cunningham, K., Müller, S., Hallberg, R. L., and Schatz, G. (1992) Cell 69, 809-822 [Medline] [Order article via Infotrieve]
  11. Glick, B. S., Wachter, C., Reid, G. A., and Schatz, G. (1993) Protein Sci. 2, 1901-1917 [Abstract/Free Full Text]
  12. Harkness, T. A. A., Nargang, F. E., van der Klei, I., Neupert, W., and Lill, R. (1994) J. Cell Biol. 124, 637-648 [Abstract]
  13. Hartl, F.-U., Ostermann, J., Guiard, B., and Neupert, W. (1987) Cell 51, 1027-1037 [Medline] [Order article via Infotrieve]
  14. Hartl, F.-U., Pfanner, N., Nicholson, D. W., and Neupert, W. (1989) Biochim. Biophys. Acta 988, 1-45 [Medline] [Order article via Infotrieve]
  15. Horwich, A. (1990) Curr. Opin. Cell Biol. 2, 625-633 [Medline] [Order article via Infotrieve]
  16. Jordi, W., Hergersberg, C., and de Kruijff, B. (1992) Eur. J. Biochem. 204, 841-846 [Abstract]
  17. Kang, P. J., Ostermann, J., Shilling, J., Neupert, W., Craig, E. A., and Pfanner, N. (1990) Nature 348, 137-143 [CrossRef][Medline] [Order article via Infotrieve]
  18. Lithgow, T., Junne, T., Wachter, C., and Schatz, G. (1994) J. Biol. Chem. 269, 15325-15330 [Abstract/Free Full Text]
  19. Martin, J., Mahlke, K., and Pfanner, N. (1991) J. Biol. Chem. 266, 18051-18057 [Abstract/Free Full Text]
  20. Miller, B. R., and Cumsky, M. G. (1991) J. Cell Biol. 112, 833-841 [Abstract]
  21. Miller, B. R., and Cumsky, M. G. (1993) J. Cell Biol. 121, 1021-1029 [Abstract]
  22. Moczko, M., Gärtner, F., and Pfanner, N. (1993) FEBS Lett. 326, 251-254 [CrossRef][Medline] [Order article via Infotrieve]
  23. Moczko, M., Ehmann, B., Gärtner, F., Hönlinger, A., Schäfer, E., and Pfanner, N. (1994) J. Biol. Chem. 269, 9045-9051 [Abstract/Free Full Text]
  24. Nicholson, D. W., Hergersberg, C., and Neupert, W. (1988) J. Biol. Chem. 263, 19034-19042 [Abstract/Free Full Text]
  25. Pfaller, R., Pfanner, N., and Neupert, W. (1989) J. Biol. Chem. 264, 34-39 [Abstract/Free Full Text]
  26. Pfanner, N., and Neupert, W. (1985) EMBO J. 4, 2819-2825 [Abstract]
  27. Pfanner, N., Craig, E. A., and Meijer, M. (1994) Trends Biochem. Sci. 19, 368-372 [CrossRef][Medline] [Order article via Infotrieve]
  28. Ramage, L., Junne, T., Hahne, K., Lithgow, T., and Schatz, G. (1993) EMBO J. 12, 4115-4123 [Abstract]
  29. Roise, D., Horvath, S. J., Tomich, J. M., Richards, J. H., and Schatz, G. (1986) EMBO J. 5, 1327-1334 [Abstract]
  30. Schneider, H., Söllner, T., Dietmeier, K., Eckerskorn, C., Lottspeich, F., Trülzsch, B., Neupert, W., and Pfanner, N. (1991) Science 254, 1659-1662 [Medline] [Order article via Infotrieve]
  31. Sims, P. J., Waggoner, A. S., Wang, C.-H., and Hoffman, J. F. (1974) Biochemistry 13, 3315-3330 [Medline] [Order article via Infotrieve]
  32. Söllner, T., Griffiths, G., Pfaller, R., Pfanner, N., and Neupert, W. (1989) Cell 59, 1061-1070 [Medline] [Order article via Infotrieve]
  33. Söllner, T., Rassow, J., and Pfanner, N. (1991) Methods Cell Biol. 34, 345-358 [Medline] [Order article via Infotrieve]
  34. Steger, H. F., Söllner, T., Kiebler, M., Dietmeier, K. A., Pfaller, R., Trülzsch, K. S., Tropschug, M., Neupert, W., and Pfanner, N. (1990) J. Cell Biol. 111, 2353-2363 [Abstract]
  35. Stuart, R. A., Gruhler, A., van der Klei, I., Guiard, B., Koll, H., and Neupert, W. (1994) Eur. J. Biochem. 220, 9-18 [Abstract]
  36. von Heijne, G. (1986) EMBO J. 5, 1335-1342 [Abstract]
  37. Voos, W., Gambill, B. D., Guiard, B., Pfanner, N., and Craig, E. A. (1993) J. Cell Biol. 123, 119-126 [Abstract]

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