N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex

The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements. The majority of these proteins are encoded in the cell's nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome. A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane. One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins. Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis. Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein. Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins. Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts). The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here.     

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.     

The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1-TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.     

Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro

Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner. Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway. The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored. The insertion of the mitochondrial DNA-encoded subunit 8 of F(1)F(0)-ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate. This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction. This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E. coli and chloroplast thylakoid membranes. (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process. (ii) It did not require protease-sensitive membrane components facing the matrix space. (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner. Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.     

Protein insertion into the inner membrane of mitochondria

The inner membrane of mitochondria harbours a large number of polypeptides, many of which have evolved from proteins of the prokaryotic progenitors of mitochondria. The sorting routes on which these proteins are integrated into the mitochondrial inner membrane reflect their phylogenetic origin: Proteins of eukaryotic descent typically reach their destination following arrest of import at the level of the inner membrane. In contrast, many proteins inherited from the prokaryotic progenitor cell are inserted into the inner membrane in an export step following translocation into the matrix. Recently, three different insertion pathways from the matrix into the inner membrane were identified which show considerable parallels to the protein insertion processes in bacteria and chloroplasts. Two of these pathways depend on the related inner membrane proteins Oxa1 and Cox18. A third route is less well defined and depends on the membrane-associated matrix protein Mba1.     

Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae

The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.     

Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1∼5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.Most mitochondrial proteins are nuclear DNA-coded, and their import into mitochondrial compartments, that is, the mitochondrial outer membrane (MOM),³ mitochondrial inner membrane (MIM), intermembrane space (IMS), and matrix, is mediated by five protein translocation systems: translocase of the outer membrane (TOM complex), sorting and assembly machinery of MOM (SAM/TOB), translocases of the inner membrane (TIM23 complex and TIM22 complex), and a fifth system in the MIM that mediates integration of proteins from the matrix into the MIM (1, 2). The last system, which has been analyzed in detail in yeast, requires a membrane potential across the MIM and matrix ATP and mediates MIM integration of the mtDNA-encoded proteins as well as the integration of certain nuclear DNA-encoded proteins considered to be of bacterial origin, such as cytochrome c oxidase subunit II, F1Fo-ATPase subunit 9, and Oxa1 (3–5). Translocation efficiency is affected by the charge difference across the transmembrane (TM) in accordance with the positive-inside rule (5). Furthermore, the matrix-exposed C-terminal segment of Oxa1 is essential for binding mitochondrial ribosomes during cotranslational integration of mtDNA-encoded proteins (6, 7). Recent reports further demonstrated that the MIM protein Mba1, as a ribosome receptor, cooperates with the C-terminal ribosome binding segment of Oxa1 (8). The machinery and the underlying mechanisms of MIM insertion from the matrix must be further analyzed.Oxa1 protein, originally identified in yeast, is a component of the matrix-to-MIM export system conserved from prokaryote to eukaryote and is involved in Oxa1 biogenesis (9–14). YidC, a bacterial homologue of Oxa1, is involved in the biogenesis of inner membrane proteins in a Sec-dependent or Sec-independent manner (15, 16). In yeast, IMS export from the matrix of the Oxa1 N-terminal segment emerging from the Tim23 channel requires a membrane potential (4, 17), and the export is compromised in mitochondria isolated from a temperature-sensitive Oxa1-expressing strain at a non-permissive temperature (12). Herrmann and Bonnefoy (18) reported that Oxa1 protein functions in the export of a single hydrophilic loop region that was artificially produced by ligating the C-terminal region of cytochrome b with cytochrome c oxidase subunit II and placed between TM segments. Direct interaction of Oxa1 with an immature subunit in complex V was observed during its biogenesis (19). So far, these studies have only been performed in yeast, and no information is available on the mechanism of topogenesis in mammals with regard to how Oxa1 is involved in the export of multiple regions in a protein molecule. Our in vivo study revealed that the correct topogenesis of Oxa1 in the MIM proceeds as a result of the cooperation of all five TMs and that the cooperation of TM1 and the species-conserved six-residue segment (GLPWWG) in the N-terminal flanking region is essential for export from the matrix of both the N-terminal segment and hydrophilic L2 into the IMS.     

The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.     

Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.

In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR). This protein attains the correct topology across the inner membrane (Nout-Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.     

Sorting pathways of mitochondrial inner membrane proteins

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa F0-ATPase has been studied as an example. F0 subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokaryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.     

Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane

Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.     

Import Pathway of Nuclear-Encoded Cytochrome c Oxidase Subunit 2 Using Yeast as a Model

Abstract: Subunit 2 of cytochrome c oxidase (Cox2) is a mitochondrial-encoded protein in most organisms. In soybean Glycine max a second Cox2 gene was identified in the nucleus which is functional, whereas the mitochondrial-encoded cox2 gene is silent. For import and sorting of the nuclear-encoded soybean Cox2 protein ( Gm Cox2p) into mitochondria, the protein has acquired an N-terminal extension of 136 amino acid residues that is cleaved off in three steps during import. To study the function and processing of the Gm Cox2p leader peptide, we used yeast as a model system. Using different leader peptide-GFP constructs, we were able to show that the i₁ intermediate is generated in the mitochondrial matrix and the mature protein is generated in the inner membrane space. Mitochondrial processing peptidase (MPP) is involved in processing the first part of the leader peptide, processing of the last part is catalysed by the inner membrane peptidase (IMP). Oxa1p is necessary for insertion of the protein into the inner mitochondrial membrane. Gm Cox2p therefore utilises many of the same components as its mitochondrial-encoded predecessor, for sorting and maturation, following its import into the mitochondria.     

A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations

Members of the Oxa1p/Alb3/YidC family mediate the insertion of various organelle or bacterial hydrophobic proteins into membranes. They present at least five transmembrane segments (TM) linked by hydrophilic domains located on both sides of the membrane. To examine how Oxa1p structure relates to its function, we have introduced point mutations and large deletions into various domains of the yeast mitochondrial protein. These mutants allowed us to show the importance of the first TM domain as well as a synergistic interaction between the first loop and the C-terminal tail, which both protrude into the matrix. These mutants also led to the isolation of a high copy suppressor, OMS1, which encodes a member of the methyltransferase family. Overexpression of OMS1 seems to increase the steady-state level of both the mutant and wild-type Oxa1p. We show that Oms1p is a mitochondrial inner membrane protein inserted independently of Oxa1p. Oms1p presents one TM and a N-in C-out topology with the C-terminal domain carrying the methyltransferase-like domain. A conserved motif within this domain is essential for the suppression of oxa1 mutations. We discuss the possible role of Oms1p on Oxa1p intermembrane space domain.     

A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.

Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Conserved mechanism of Oxa1 insertion into the mitochondrial inner membrane

Oxa1 is the mitochondrial representative of a family of related proteins that mediate the insertion of substrate proteins into the membranes of bacteria, chloroplasts, and mitochondria. Several studies have demonstrated that the bacterial homologue YidC participates both in the direct uptake of proteins from the bacterial cytosol, and in the uptake of nascent proteins from the Sec translocase. Studies on the biogenesis of membrane proteins in mitochondria established that Oxa1 has the capability to receive substrates at the inner surface of the inner membrane. In this study, we asked if Oxa1 may similarly cooperate with a protein translocase within the membrane. Since Oxa1 is involved in its own biogenesis, we used the precursor of Oxa1 as a model protein and investigated its import pathway. We found that immediately after import into mitochondria, Oxa1 initially accumulates at Tim23 that forms the inner membrane protein translocase. Cleavage of the Oxa1 presequence is dependent on mtHsp70, a heat shock protein of the mitochondrial matrix. However, mutant mtHsp70 showing a defect in the release of bound substrate proteins does not interfere with subsequent membrane insertion, indicating that membrane insertion of the mature protein is essentially mtHsp70-independent. We conclude that Oxa1 has the ability to accept preproteins within the membrane.     

The Oxa1 protein forms a homooligomeric complex and is an essential part of the mitochondrial export translocase in Neurospora crassa

The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. We have identified the Neurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue. Despite the low level of identity, the function of the homologues is conserved as the N. crassa gene fully complemented a yeast null mutant. Genetic analysis revealed that Oxa1 is essential for viability in N. crassa. Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV. Isolation of the Oxa1 complex from N. crassa mitochondria revealed a 170-180-kDa complex that contained exclusively Oxa1. Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits.     

Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator.

Import of precursor proteins across the mitochondrial inner membrane requires ATP in the matrix. However, some precursors can still cross the outer membrane in ATP-depleted mitochondria. Here we show that the adenine nucleotide translocator is imported normally into the inner membrane after the matrix has been depleted of ATP. This result supports the earlier suggestion that the translocator inserts into the inner membrane without passing through the matrix. Depletion of matrix ATP also has no detectable effect on the import and maturation of cytochrome c1, which is targeted to the intermembrane space. It thus seems probable that cytochrome c1 does not completely cross the inner membrane during its import pathway.     

Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.