In the yeast Saccharomyces cerevisiae only eight structural proteins are encoded by the mitochondrial genome (Mason and Schatz, 1973; Borst and Grivell, 1978; Tzagoloff and Meyers, 1986). The synthesis of these gene products, all integral inner membrane proteins with the exception of the ribosomal Var1 protein, has been proposed to occur concomitantly with the insertion into and translocation across the membrane. Evidence for this coupled mechanism comes from a number of independent observations. Ribosomes undergoing the synthesis of these proteins are found associated with the inner boundary membrane (André, 1965; Vignais et al., 1969; Watson, 1972). The interaction between the ribosomes and the inner membrane has been reported to be mediated by specific proteinaceous components that bind directly either to the ribosome or to the translated mRNA (Constanzo and Fox, 1990; Michaelis et al., 1991). In addition nascent polypeptide chains released from mitochondrial ribosomes with puromycin treatment are present in the inner membrane and are resistant to extraction at alkaline pH (Poyton et al., 1992; Pajic et al., 1994). Finally, no detectable pools of these proteins have been observed to be soluble in the matrix (Severino and Poyton, 1980; Fujiki et al., 1982; McKee and Poyton, 1984). Whether this linkage between synthesis and membrane insertion is obligatory and thus functional or whether it represents a kinetic phenomenon has not yet been clarified.

All of the membrane proteins made on mitochondrial ribosomes become inserted into the inner membrane in a manner that requires the complete translocation of hydrophilic charged segments across the lipid bilayer to the intermembrane space. Information on the mechanisms, energetic requirements, or components involved in these processes is scarce. A number of specialized factors involved in post-translational events in the assembly pathways of these proteins have been identified during the past years. These factors, such as Sco1p, ABC1, ATPase10, COX10, ()and COX11, display a strict specificity for certain complexes and appear to operate at the later stage of assembly rather than at membrane translocation (Krummeck and Rödel, 1990; Ackerman and Tzagoloff, 1990; Nobrega et al., 1990; Tzagoloff et al., 1990; Bousquet et al., 1991). No general component required for insertion and translocation of all these gene products (i.e. a translocation machinery) has been identified to date. In addition very little is known about the energetic or other requirements of protein export from the matrix, besides one study that showed that maturation of COXII is partially dependent on , but it remained unclear whether export or a post-translocational step was affected (Clarkson and Poyton, 1989; Poyton et al., 1992).

We have addressed questions concerning the process of sorting and assembly of mitochondrial gene products using cytochrome oxidase subunit II (COXII) as a model system, for the following reasons. First, the COXII protein has an established and relatively simple topology; a two-membrane-spanning protein with both N- and C-terminal segments exposed to the intermembrane space (Bisson et al., 1982). The N-terminal tail of mature COXII is relatively short (27 amino acids in the case of S. cerevisiae), and the C-terminal domain is considerably longer (144 amino acids). Both termini are very hydrophilic with a strong net negative charge that must become translocated across the membrane following synthesis in the matrix. Second, COXII is synthesized as a precursor, preCOXII, that contains an N-terminal presequence (15 amino acid residues in S. cerevisiae and 12 in Neurospora crassa). PreCOXII is processed by the Imp1p protease on the external surface of the inner membrane. Hence the processing to its mature size is a convenient measure of the translocation of the N terminus across the inner membrane, where it gains access to the Imp1p protease. Finally, the accessibility of the large C-terminal acidic domain in the intermembrane space to exogenously added protease after opening of the outer membrane can be used as a criterion for insertion of the second transmembrane domain leading to export of the C-terminal segment of the protein.

In this report we present evidence that the sorting of COXII can be experimentally dissected into a number of distinct stages. The process of translation of COXII can be separated from subsequent translocational events, demonstrating that co-translocational translation is not obligatory. Furthermore the stable insertion of the transmembrane domains into the membrane is supported by a membrane potential across the inner membrane. The export step of COXII is independent of maturation by the Imp1p protease and of the assembly into the cytochrome c oxidase complex. Finally, when fused to a mitochondrial presequence, the N-terminal portion of preCOXII can be imported into the mitochondrial matrix. Upon accumulation in the matrix, this species can be correctly sorted, leading to export of its N-terminal tail and Imp1p processing in the intermembrane space. Similarities of this export event to bacterial protein export provide support for a conservation of the mechanisms of sorting from prokaryotes to eukaryotic organelles.

MATERIALS AND METHODS

Yeast Strains

Mutagenesis of the preCOXII Gene and Creation of pSu9(1-66)preCOXII-DHFR Fusion Protein

Isolation of Mitochondria

Import of Precursor Proteins into Mitochondria

Labeling of Mitochondrial Translation Products

Antibody Production and Immunoprecipitation

For immunoprecipitation of COXII, following in vitro labeling, mitochondria were reisolated, washed in washing buffer, and lysed for 10 min at 4 °C in 10 µl of 1% SDS, 1 mM phenylmethylsulfonyl fluoride. After an incubation for 2 min at 96 °C, samples were diluted with 1 ml of 1% Triton X-100 lysis buffer (1% Triton X-100 (w/v), 300 mM NaCl, 10 mM Tris/HCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4). Immunoprecipitation of COXII was then performed using either the N- or C-terminal specific antisera, as indicated and according to published procedures (Nicholson, et al., 1987).

Complex formation with mt-Hsp70 was analyzed by co-immunoprecipitation using an antibody specific for the Ssc1p, the mt-Hsp70 protein as described before (Herrmann et al., 1994b).

Sucrose Gradient Centrifugation Analysis of the Cytochrome Oxidase Complex

Other Procedures

RESULTS

Sorting of COXII Requires a Membrane Potential across the Inner Membrane

Following translation in energized mitochondria, COXII accumulated as its mature size form, indicating that efficient export of the N terminus of COXII had taken place (Fig. 1A, lane 1). When the intermembrane space was opened the mature COXII was largely resistant to added protease (Fig. 1A, lane 2). Mature size COXII could be immunoprecipitated with peptide-specific antibodies raised against the N and C termini (Fig. 1, B and C, lanes 1). Although the C terminus of this mature size COXII species could not be degraded by added protease, it was exported (N-C) and folded. This is documented by the following results. (i) When protein synthesis was in mitoplasts under similar energetic conditions, the mature COXII was sensitive to exogenously added proteases, as it does not appear to fold to a resistant conformation (cf. Fig. 3). (ii) When synthesized in mitochondria in the absence of a membrane potential, translocation of the C terminus of the newly synthesized mature COXII is inhibited, and protease treatment resulted in the generation of a C-terminal 31-kDa fragment protected in the mitochondrial matrix (see below). Thus the lack of production of such a protease-protected C-terminal fragment in mitoplasts when synthesized in energized mitochondria indicates that export of this domain had occurred. (iii) Endogenous COXII was also largely resistant to proteinase K attack from the intermembrane space as demonstrated by immunoblotting (results not shown).

Figure 1: Precursor of COXII accumulates after depletion of the membrane potential. Mitochondrial translation was monitored in vitro with [S]methionine in the presence of 1 µM valinomycin, 20 µM CCCP, 10 mM NaN, 10 mM KCN, 10 µM nigericin or in translation buffer alone, as indicated, for 20 min at 25 °C. Mitochondria then were either directly resuspended in SDS sample buffer (A), or converted to mitoplasts in the presence of proteinase K (100 µg/ml). Resulting mitoplasts were either directly solubilized in sample buffer (A, swelling +PK) or lysed in detergent used for immunoprecipitations with antiserum specific for either the N terminus (B) or the C terminus of COXII (C), as described under ``Materials and Methods.'' All samples were analyzed by SDS-PAGE and fluorography. The resulting films were quantified by laser densitometry. The levels of proteinase K-resistant preCOXII (N-C) and of the C-terminal (31-kDa) fragment (N-C) are expressed as a percentage of the total labeled COXII in intact mitochondria (D). val, valinomycin; nig, nigericin; coxI, coxII, and coxIII, subunits I, II, and III of the cytochrome oxidase complex, respectively; cyt b, cytochrome b.

Figure 3: Sorting of COXII is independent of assembly of the COX complex. A, protein synthesis in wild-type (lanes 1-4), coxIV (lanes 5 and 6), mit (lanes 7 and 8), and GR20 mitochondria (lanes 9 and 10) was performed as described in Fig. 1. Mitochondria were swollen and treated with proteinase K (lanes 2-10), and lysed in 1% Triton X-100 buffer. The extracts were used for immunoprecipitation with antisera against the N terminus (lanes 3, 5, 7, and 9) and the C terminus of COXII (lanes 4, 6, 8, and 10). B, following translation in the presence of [S]methionine for 60 min at 25 °C, mitochondria (100 µg) were reisolated, washed, and lysed with deoxycholate. Detergent extract was loaded onto a 5-20% sucrose density gradient and subjected to centrifugation, as described under ``Materials and Methods.'' Fractions were collected, precipitated with trichloroacetic acid, analyzed by SDS-PAGE, and transferred on nitrocellulose. The blot was decorated with antisera against COXII, Mge1p, and cytochrome b (tetramer, 250 kDa). The signals of endogenous () and newly synthesized () COXII were quantified by laser densitometry. The resulting distribution is shown. The positions of the Mge1p and cytochrome b (cyt b) marker proteins are indicated by arrows.

Depletion of the membrane potential across the inner membrane by the addition of valinomycin resulted in the accumulation of preCOXII (Fig. 1A, lane 3). This precursor was largely resistant to digestion by protease under hypotonic swelling conditions (N-C topology) (Fig. 1A, lane 4). In contrast, the mature size COXII accumulated in deenergized mitochondria was sensitive to the proteinase K. The degradation of the COXII species gave rise to the above mentioned fragment, which migrated slightly faster than the cytochrome b protein and was found protected in mitoplasts (Fig. 1A, lane 4). This 31-kDa fragment could be immunoprecipitated with the antibody specific for the C terminus (Fig. 1C, lane 2) and not with the N-terminal one (Fig. 1B, lane 2). The size of this fragment is in good agreement with that expected following proteolytic removal of the complete exported N terminus of the COXII. The protease protection of such a C-terminal fragment arises from the inhibition of export of the C-terminal domain of COXII (N-C topology). The presence of CCCP, azide, cyanide, and nigericin had a similar inhibitory effect on the translocation of both the N- and C-terminal tails (Fig. 1A, lanes 5-12, and Fig. 1B, and C, lanes 3-6). Quantification of these data showed that the translocation of the C terminus was more dependent on a membrane potential than that of the N terminus (Fig. 1D).

We analyzed the insertion of the transmembrane domains of the newly synthesized COXII into the inner membrane by the resistance to extraction at alkaline pH (Table 1). Following translation in energized wild-type mitochondria, accumulated mature COXII behaved as an integral membrane protein; it was not extracted at alkaline pH. In the absence of a membrane potential, accumulated preCOXII was recovered in the soluble fraction following carbonate treatment. PreCOXII accumulated in mitochondria bearing a defective Imp1p protease was not extractable at alkaline pH when synthesized in the presence of a membrane potential (Table 1). Therefore, the extractability at alkaline pH of the precursor form of COXII accumulated in deenergized mitochondria reflects a lack of insertion into the inner membrane. Failure to do so prevents export of N and C termini and hence results in the accumulation of preCOXII in the matrix. Furthermore accumulation of a nontranslocated form of preCOXII demonstrates that the co-translocational translation of mitochondrial encoded membrane proteins is not obligatory.

COXII Can Be Correctly Sorted in Mitoplasts but Fails to Attain to a Protease-resistant Conformation

Figure 2: Export of COXII following synthesis in mitoplasts. Mitochondria were either mock-treated (lanes 1 and 2) or were converted to mitoplasts by hypotonic swelling (lanes 3-8) for 20 min on ice. Mitoplasts (MP) or mitochondria (M), respectively, were reisolated, resuspended in translation buffer, and preincubated for 5 min at 25 °C in the absence (+, lanes 1-4) or in the presence of 1 µM valinomycin (-, lanes 5-8). Labeling was carried out as described in Fig. 1for 20 min at 25 °C. All samples were divided and were either treated with proteinase K (lanes 2, 4, 6, and 8) (under swelling conditions in the case of mitochondria; lane 2) or were mock-treated (lanes 1, 3, 5, and 7). Samples were subjected to centrifugation, and membrane pellets were lysed in SDS-sample buffer and analyzed by SDS-PAGE. Radiolabeled proteins were visualized by fluorography (A), and resulting films were quantified. The levels of preCOXII (pCOXII) and mature COXII (mCOXII) before and after protease treatment are shown (B). f indicates the mobility of the 31-kDa C-terminal COXII fragment.

In summary these results suggest that soluble components or co-factors of the intermembrane space are not required to mediate the export of COXII. They may, however, be required for the acquisition of protease resistance following the membrane translocation events.

Sorting of COXII across the Inner Membrane Is Independent of Assembly into the COX Complex

Following translation in all of these different mitochondria, newly synthesized COXII was correctly processed to its mature species, indicating that export of the N terminus had occurred (Fig. 3A). No accumulation of the C-terminal fragment characteristic of inhibition of export was observed in these mitochondria when they were subjected to hypotonic swelling in the presence of protease. We conclude, therefore, that the export process of preCOXII is independent of its assembly into the COX complex.

We further tested whether following translation in wild-type mitochondria, newly synthesized COXII was assembled into the COX complex (Fig. 3B). After labeling of translation products, a detergent extract of the mitochondria was subjected to sucrose gradient centrifugation. Endogenous COX complex peaked in fractions 5 and 6. This corresponded to a complex of 250 kDa, the expected size for the COX complex (Fig. 3B). The newly synthesized radiolabeled COXII, on the other hand, was detected at the top of the gradient. A similar observation was made when the detergent extract was subjected to gel filtration chromatography (results not shown). In conclusion, the newly synthesized radiolabeled mature size COXII did not assemble into the COX complex, indicating that export and assembly are two distinct events.

Import and Sorting of pSu9(1-66)preCOXII(1-74)-DHFR

Figure 4: Import, sorting, and degradation of pSu9(1-66)preCOXII(1-74)-DHFR is dependent on the membrane potential and ATP. A, the scheme shows the preCOXII gene and the pSu9(1-66)preCOXII(1-74)-DHFR construct. The positions of the cleavage sites of the mitochondrial matrix protease (MPP) present in the Su9 presequence and of the intermembrane space-localized protease (Imp1p) cleavage site of the preCOXII presequence as well as the transmembrane domain (TM1) present in the mature part of COXII are indicated. B, radiolabeled pSu9(1-66)preCOXII(1-74)-DHFR was imported into isolated mitochondria either in the absence (lanes 1-3) or in the presence of 2 mM ATP (lanes 4-6), 2 mM NADH (lanes 7-9), or 2 mM NADH plus 2 mM ATP (lanes 10-12) for 20 min at 25 °C. Each sample was divided into three aliquots and was mock-treated (lanes 1, 4, 7, and 10), treated with proteinase K (lanes 2, 5, 8, and 11), or converted to mitoplasts in the presence of proteinase K (lanes 3, 6, 9, and 12). Samples were subsequently analyzed by SDS-PAGE, transferred onto nitrocellulose, and visualized by autoradiography. Blots were immunodecorated with antisera against cytochrome c oxidase (CCPO) and the mitochondrial GrpE-homologue Mge1 (MGE), as markers for the intermembrane space and matrix, respectively. pSu9, pSu9(1-66)preCOXII(1-74)-DHFR; pCOXII, preCOXII(1-74)-DHFR; mCOXII, mature, Imp1p-processed COXII(1-74)-DHFR; PK, proteinase K.

In the presence of added NADH, the N terminus of preCOXII(1-74)-DHFR was exported back across the inner membrane, as the COXII presequence became cleaved by the Imp1p protease (Fig. 4B, lanes 7 and 8) and the N-terminal tail was accessible to added protease under hypotonic swelling conditions (N-C topology) (Fig. 4B, lane 9). This export event was only observed in the presence of added NADH, suggesting that the level of membrane potential required for the export process was higher than that necessary for the initial import step. In the Imp1p-defective mitochondria, processing of preCOXII(1-74)-DHFR to its mature size form was not observed, but the N terminus became exported and exposed to the intermembrane space (results not shown). Thus, in agreement with what was observed for its mitochondrially synthesized counterpart, export of the N terminus of preCOXII and processing by Imp1p are two independent steps.

In the presence of added ATP, pSu9(1-66)preCOXII(1-74)-DHFR was imported into the mitochondria, where it accumulated as preCOXII(1-74)-DHFR in the mitochondrial matrix (Fig. 4B, lane 6). Export to the intermembrane space was not observed. This result demonstrates that the membrane potential requirements are directly for export and do not reflect an indirect requirement for matrix ATP synthesis. In the presence of both NADH and ATP, efficient export of the N-terminal tail occurred (Fig. 4B, lane 12). In addition, proteolytic degradation to a number of smaller fragments was also observed. Apparently once inserted into the membrane this fusion protein becomes a substrate for an ATP-dependent protease. As proteolytic degradation in the absence of membrane insertion (i.e. +ATP, no added NADH) was not observed (Fig. 4B, lane 6), membrane insertion precedes proteolytic degradation.

In summary, the N terminus of COXII can be imported into mitochondria in a post-translational manner, delivering it to the matrix, the site of synthesis of its mitochondrially encoded counterpart. This imported form of COXII can embark on an export event across the inner membrane, where it becomes processed by Imp1p protease.

The Import and Export Steps Are Two Independent Events

Figure 5: Kinetics of import, sorting, interaction with mt-Hsp70, and folding of pSu9(1-66)preCOXII(1-74)DHFR in the presence of NADH. Radiolabeled pSu9(1-66)preCOXII(1-74)-DHFR was imported into isolated mitochondria in the presence of 2 mM NADH and 0.2 mM ATP for 2 min at 25 °C and then trypsin-treated. Following the addition of soybean trypsin inhibitor, samples were then either left on ice (lanes 1) or incubated further (2. incubation) at 25 °C (lanes 2-4) for the times indicated. Samples were divided into three parts; one was mock-treated (A, mitochondria, M), and another was converted to mitoplasts in the presence proteinase K (panel B, MP + PK). The mitochondria from the third aliquot were lysed in Triton X-100 buffer, and the extracts were either co-immunoprecipitated with antisera against mt-Hsp70 (C) or preimmune serum (p.i.) (D) or were treated with proteinase K and trichloroacetic acid-precipitated to assay the amount of folded DHFR (results not shown), as described under ``Materials and Methods.'' iSu9COXIIDHFR, intermediate mitochondrial matrix protease-processed Su9(1-66)preCOXII(1-74)-DHFR; pCOXIIDHFR, preCOXII(1-74)-DHFR; mCOXII-DHFR, Imp1p-processed COXII(1-74)- DHFR.

Both the intermediate form of Su9(1-66)preCOXII(1-74)-DHFR (processed only once by mitochondrial matrix protease, residues 1-35 removed) and the preCOXII(1-74)-DHFR species (residues 1-66 removed), which accumulated in the matrix after early time points of import were present in a complex with mt-Hsp70 as shown by co-immunoprecipitation (Fig. 5C, lanes 1 and 2). The DHFR domain of these imported species was tightly folded (results not shown), suggesting that the interaction of mt-Hsp70 had occurred with the Su9 and/or the preCOXII part of the protein. Chase to the exported form was accompanied by a release from mt-Hsp70 (Fig. 5C, lanes 3 and 4). It is tempting to speculate that mt-Hsp70, by binding to these domains of the protein, prevents its subsequent aggregation and maintains the protein in a competent conformation necessary for the further membrane translocation event.

The chase of matrix-localized form to the exported one requires an energized inner membrane, as is shown by the following experiment (Fig. 6A). PSu9(1-66)preCOXII(1-74)-DHFR was initially imported into mitochondria in the absence of added NADH. Following trypsin treatment, chase of the matrix-accumulated species to the exported form and its subsequent maturation by Imp1p protease were related to the NADH concentration (Fig. 6A). The presence of inhibitors of the membrane potential inhibited this chase reaction (Fig. 6B). Interestingly this chase of the preCOXII(1-74)-DHFR species to an N-C topology displayed a similar energetic requirement as the export of the N terminus of the mitochondrially encoded COXII species. CCCP and azide were more effective inhibitors than valinomycin. Export was, however, only weakly inhibited by prior depletion of the matrix of ATP (Fig. 6B). As this matrix-localized species was no longer complexed to mt-Hsp70 after the first incubation (results not shown), matrix ATP appears only to be required for release from mt-Hsp70 and not at later stages of the export of the N-terminal tail.

Figure 6: Export of preCOXII(1-74)-DHFR is strongly dependent on a membrane potential. pSu9(1-66)preCOXII(1-74)-DHFR was imported into mitochondria in the absence of added NADH for 5 min at 25 °C. Following trypsin treatment, mitochondria were further incubated for 20 min at 25 °C either in the presence of increasing concentrations of NADH (A) or in the presence of 2 mM NADH together with either no further additions, or 1 µM valinomycin, 20 µM CCCP, 1 mM azide, or 20 µM oligomycin 40 units/ml apyrase, as indicated (B). Then the samples were divided; one-half of each was subjected to swelling in the presence of proteinase K, and the other half was mock-treated. Samples were analyzed by SDS-PAGE and fluorography, and the resulting films were then quantified. The levels of exported COXII(1-74)-DHFR (N terminus protease accessible in mitoplasts) () and Imp1p-matured COXII(1-74)-DHFR () are shown as a percentage of total imported (trypsin-resistant) pSu9(1-66)preCOXII(1-74)-DHFR. COXIIDHFR, COXII(1-74)-DHFR.

In summary we demonstrate that a fusion protein encompassing the first transmembrane domain of preCOXII can be imported into mitochondria. This protein does not become arrested at the level of the inner membrane upon import in a stop-transfer manner but is completely imported into the matrix despite its hydrophobicity. Upon accumulation in the matrix this protein has the ability to access and embark on an export pathway very similar or identical to that of its mitochondrially encoded counterpart. It becomes exported in a membrane potential-dependent manner across the inner membrane, where it undergoes processing by the Imp1p peptidase. The import and export steps can be dissected from each other due to the strict dependence of the export process on a high membrane potential requirement.

DISCUSSION

This study provides novel insights into the mechanism of inner membrane insertion and translocation of mitochondrially encoded proteins. We have characterized this process using cytochrome oxidase subunit II (COXII) as a model protein (Fig. 7). COXII is synthesized as a precursor, preCOXII, in the mitochondrial matrix and undergoes an insertion into and across the inner membrane, which results in the complete translocation of both the N and C termini across the membrane into the intermembrane space (N-C topology). The process of export of these hydrophilic domains has been experimentally dissected here (Fig. 7). In the absence of a proton motive force (µH) across the inner membrane, correct sorting of newly synthesized COXII is inhibited. In this case accumulation of a matrix-localized preCOXII (N-C topology) and of a mature size species whose C-terminal domain remained in the matrix (N-C) were observed. Thus we conclude that the translocation of both termini is supported by a membrane potential. The translocation of the C-terminal domain displayed a requirement for a higher membrane potential. Whether this is because the C terminus is significantly longer or more negatively charged than the N terminus, or a combination of both, awaits further investigation. Accumulation of preCOXII in the absence of a µH as non-membrane-integrated species, speaks for a requirement of the membrane potential also at the step of insertion of the transmembrane domains of COXII into the inner membrane. Both the and pH components of the µH appear to be supporting the export process, as all inhibitors tested interfered with the translocation of both termini. Thus we conclude the membrane potential does not directly influence the processing of preCOXII to the mature species by Imp1p, but rather affects the preceding step of export from the matrix.

Figure 7: Working model for the sorting of the N and C terminus of COXII into the intermembrane space after synthesis in the mitochondrial matrix. IM, inner membrane; IMS, intermembrane space; PS, presequence; TM1, transmembrane domain 1; TM2, transmembrane domain 2.

Correct sorting of the N terminus of COXII was observed when a COXII-DHFR chimeric protein was imported into the mitochondria, a process facilitated by a matrix-targeting signal fused in front of the COXII sequence. The entire protein was accumulated in the matrix and then could be exported to an N-C topology, probably along the same sorting pathway as the authentic COXII, as demonstrated by Imp1p processing and similar energetic requirements. In this respect it is interesting to note that in leguminous plants COXII is encoded in the nucleus. The gene encodes a mitochondrial targeting signal that is separated from the COXII open reading frame by an intron (Nugent and Palmer, 1991; Covello and Gray, 1992). Thus in these plants sorting of COXII in a post-translational manner is obligatory. Together these observations substantiate the conclusion that the process of mitochondrial protein export is not necessarily coupled to protein synthesis. Since, however, pools of mitochondrially synthesized membrane proteins in the matrix are normally not observed, both processes are probably closely coordinated under physiological conditions. A tight control of translation and translocation could serve to enhance both the kinetics and efficiency of export.

Export of the N terminus of COXII was found to occur independently of export of the C terminus. How far the insertion of the first transmembrane domain is coupled to that of the cleavable presequence of COXII is not clear presently. The function of this presequence is unknown; interestingly, it is not present on all COXII proteins sequenced, e.g. bovine and human (Stef-fans et al., 1979; Chomyn et al., 1981). Preliminary results have shown that in the absence of the transmembrane domain, translocation of the N-terminal domain into the intermembrane space and Imp1p processing can occur, but inefficiently. ()Thus the presequence together with the N-terminal tail may have some targeting function; however, it is clearly enhanced when the transmembrane domain is present, which may function to stabilize the protein in the lipid bilayer.

In addition export of COXII was observed to occur independently of its assembly into a functional COX complex. A similar observation was also made for COXI and COXIII (results not shown). Thus these newly synthesized proteins are not directly inserted from the matrix into their final functional locations. As this process of export requires the translocation of sometimes highly charged domains, this event may be mediated by a specific channel in the inner membrane. The question of how such a putative channel is composed and whether it is used by all the mitochondrially encoded membrane proteins awaits further analysis. A possible candidate for a component of such a channel could be the recently described OXA1/pet1402 gene product, a multispanning inner membrane protein. Deletion of this gene or mutations in it, result in a petite phenotype and interestingly in accumulation of uncleaved preCOXII (Bauer et al., 1994; Bonnefoy et al., 1994).

Finally, several aspects of the COXII sorting process resemble protein export in prokaryotes. Export of both termini of mitochondrial COXII requires a membrane potential across the inner membrane. In bacteria a number of reports have demonstrated that the export of both N- and C-terminal domains are supported by a µH across the plasma membrane (Schiebel et al., 1991; Whitley et al., 1994). Furthermore, both the exported N and C tails of mitochondrial and bacterial COXII are negatively charged, while the matrix loop (i.e. nontranslocated segment) between the two transmembrane domains is positively charged. As shown in the bacterial system, positively charged amino acids flanking transmembrane segments tend to be more prevalent in the cytoplasmic than in periplasmic space (``positive-inside'' rule) (von Heijne, 1989; Boyd and Beckwith, 1990; Dalbey, 1990). We propose that these positive charges flanking the transmembrane domains of mitochondrial COXII serve to retain this segment in the matrix and thereby determine the orientation of the membrane insertion process. Moreover, the Imp1p protease responsible for the maturation of preCOXII is homologous to the bacterial leader peptidase. The similarity of these two proteases indicates conservation of at least one component of the bacterial secretory machinery during the evolution of mitochondria from their prokaryotic ancestors. Most importantly we demonstrate here that a COXII-DHFR derivative, when imported into mitochondria, accumulates initially in the matrix in such a manner that it is competent to embark on this export pathway. Thus we show that a bacterial type of export pathway exists in mitochondria, and this pathway can be accessed by nuclearly encoded proteins following their import into the mitochondrial matrix.

FOOTNOTES

*

This work was supported by grants from the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 184 (Teilprojekt B2) and from the Münchener Medizinische Wochenschrift (to R. A. S.). 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: Boehringer Mannheim GmbH, Werk Penzberg, Nonnenwald 2, 82377 Penzberg, Germany.

¶

Present address: Dept. of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.

()

The abbreviations used are: COX, cytochrome c oxidase; COXI, COXII, etc., COX subunit I, II, etc.; preCOXII, COXII precursor; CCCP, carbonyl cyanide m-chlorophenylhydrazone; mt-Hsp70, mitochondrial heat shock protein 70; Su9, subunit 9; DHFR, dihydrofolate reductase; PAGE, polyacrylamide gel electrophoresis.

()

J. M. Herrmann, unpublished results.

ACKNOWLEDGEMENTS

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