Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub- mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.     

Mitochondrial protein import

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.     

Transport of proteins to the mitochondrial intermembrane space: the ''sorting'' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.     

In vitro import of cytochrome c peroxidase into the intermembrane space: release of the processed form by intact mitochondria

Cytochrome c peroxidase (CCP) is a nuclearly encoded hemoprotein located in the intermembrane space (IMS) of Saccharomyces cerevisiae mitochondria. Wild-type preCCP synthesized in rabbit reticulocyte lysates, however, was inefficiently translocated into isolated mitochondria and was inherently resistant to externally added proteases. To test whether premature heme addition to the apoprecursor was responsible for the protease resistance and the inability to import preCCP, site-directed mutagenesis was used to replace the axial heme ligand (His175) involved in forming a pseudo-covalent link between the heme iron and CCP. Mutant proteins containing Leu, Arg, Met, or Pro at residue 175 of mature CCP were sensitive to proteolysis and were imported into isolated mitochondria as judged by proteolytic processing of the precursor. The inhibition of wild-type CCP translocation across the outer membrane may result from the inability of the heme-containing protein to unfold during the translocation process. Although the protease responsible for cleaving preCCP to its mature form is believed to be located in the IMS, most of the processed CCP was located in the supernatant rather than the mitochondrial pellet. Since the outer membranes were shown to be intact, the anomalous localization indicated that preCCP may not have been completely translocated into the IMS before proteolytic processing or that conformationally labile proteins may not be retained by the outer membrane. Proteolytic maturation of preCCP also occurred in the presence of valinomycin, suggesting that the precursor may be completely or partially translocated across the outer mitochondrial membrane independent of a potential across the inner mitochondrial membrane.     

Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria

The import of cytochrome c into Neurospora crassa mitochondria was examined at distinct stages in vitro. The precursor protein, apocytochrome c, binds to mitochondria with high affinity and specificity but is not transported completely across the outer membrane in the absence of conversion to holocytochrome c. The bound apocytochrome c is accessible to externally added proteases but at the same time penetrates far enough through the outer membrane to interact with cytochrome c heme lyase. Formation of a complex in which apocytochrome c and cytochrome c heme lyase participate represents the rate-limiting step of cytochrome c import. Conversion from the bound state to holocytochrome c, on the other hand, occurs 10-30-fold faster. Association of apocytochrome c with cytochrome c heme lyase also takes place after solubilizing mitochondria with detergent. We conclude that the bound apocytochrome c, spanning the outer membrane, forms a complex with cytochrome c heme lyase from which it can react further to be converted to holocytochrome c and be translocated completely into the intermembrane space.     

Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space.

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.     

The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane

A subset of mitochondrial carrier proteins from plants contain a cleavable N-terminal extension. We have used a reconstituted protein import assay system into intermembrane space-depleted mitochondria to study the role of the cleavable extension in the carrier import pathway. Insertion of carrier proteins into the inner membrane can be stimulated by the addition of a soluble intermembrane space fraction isolated from plant mitochondria. Greater stimulation of import of the adenine nucleotide carrier (ANT) and phosphate carrier (Pic), which contain N-terminal cleavable extensions, was observed compared to the import of the oxoglutarate malate carrier (OMT), which does not contain a cleavable extension. Removal of the N-terminal cleavable extension from ANT and Pic resulted in loss of stimulation of insertion into the inner membrane. Conversely, addition of the N-terminal extension from ANT or Pic to OMT resulted in significantly enhanced insertion into the inner membrane. The polytopic inner membrane proteins TIM17 and TIM23 that are imported via the carrier import pathway contain no cleavable extension, displayed high-level stimulation of insertion into the inner membrane by addition of the intermembrane space fraction. Addition of the N-terminal cleavable extension from carrier proteins to TIM23 enhanced insertion of TIM23 into the inner membrane even in the absence of the soluble intermembrane space fraction. Together, these results demonstrate that the cleavable N-terminal extensions present on carrier proteins from plants are required for efficient insertion into the inner mitochondrial membrane, and that they can stimulate insertion of any carrier-like protein into the inner membrane.     

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.     

Biogenesis of cytochrome c1. Role of cytochrome c1 heme lyase and of the two proteolytic processing steps during import into mitochondria

The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.     

Cyc2p, a membrane-bound flavoprotein involved in the maturation of mitochondrial c-type cytochromes

Mitochondrial apocytochrome c and c1 are converted to their holoforms in the intermembrane space by attachment of heme to the cysteines of the CXXCH motif through the activity of assembly factors cytochrome c heme lyase and cytochrome c1 heme lyase (CCHL and CC1HL). The maintenance of apocytochrome sulfhydryls and heme substrates in a reduced state is critical for the ligation of heme. Factors that control the redox chemistry of the heme attachment reaction to apocytochrome c are known in bacteria and plastids but not in mitochondria. We have explored the function of Cyc2p, a candidate redox cytochrome c assembly component in yeast mitochondria. We show that Cyc2p is required for the activity of CCHL toward apocytochrome c and c1 and becomes essential for the heme attachment to apocytochrome c1 carrying a CAPCH instead of CAACH heme binding site. A redox function for Cyc2p in the heme lyase reaction is suggested from 1) the presence of a noncovalently bound FAD molecule in the C-terminal domain of Cyc2p, 2) the localization of Cyc2p in the inner membrane with the FAD binding domain exposed to the intermembrane space, and 3) the ability of recombinant Cyc2p to carry the NADPH-dependent reduction of ferricyanide. We postulate that, in vivo, Cyc2p interacts with CCHL and is involved in the reduction of heme prior to its ligation to apocytochrome c by CCHL.     

The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes

The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane beta-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.     

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.

The pathway by which cytochromes c1 and b2 reach the mitochondrial intermembrane space has been controversial. According to the "conservative sorting" hypothesis, these proteins are first imported across both outer and inner membranes into the matrix, and then are retranslocated across the inner membrane. Our data argue against this model: import intermediates of cytochromes c1 and b2 were found only outside the inner membrane; maturation of these proteins was independent of the matrix-localized hsp60 chaperone; and dihydrofolate reductase linked to the presequence of either cytochrome was imported to the intermembrane space in the absence of ATP. We conclude that cytochromes c1 and b2 are sorted by a mechanism in which translocation through the inner membrane is arrested by a "stop-transfer" signal in the presequence. The arrested intermediates may be associated with a proteinaceous channel in the inner membrane.     

Diverse mechanisms and machineries for import of mitochondrial proteins

Mitochondria are ubiquitous organelles that play an essential role in energy conversion and biosynthetic pathways in eukaryotic cells. Most mitochondrial proteins must be imported from the cytosol and sorted into one of the four mitochondrial subcompartments, the outer membrane, the intermembrane space, the inner membrane and the matrix. Studies in recent years revealed a remarkable diversity of mechanisms and machineries that are required for the import of proteins into mitochondria. At least four different pathways for the sorting and assembly of nuclear-encoded mitochondrial proteins have been identified.     

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.     

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.     

The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70.

Cytochrome b2 is imported into mitochondria and sorted to the intermembrane space by a bipartite N-terminal presequence, which is a matrix targeting sequenced followed by an intermembrane space sorting signal. The N-terminus of the mature protein forms a folded heme binding domain that depends on the unfoldase function of matrix (mt) Hsp70 for import. We report that the distance between the presequence and the heme binding domain is critical for the ability of mt-Hsp70 to promote import of the domain. Hybrid proteins with 40 or more amino acids between the presequence and the heme binding domain are arrested in the import machinery. The translocation arrest can be overcome by unfolding of the preprotein or by inactivation of the intermembrane space sorting signal. Moreover, the sorting signal prevents backsliding of the precursor polypeptide in the import site in the initial import step, when the signal has not made contact with the matrix. The results indicate that the sorting signal interacts with component(s) of the inner membrane/intermembrane space during the initial import step and promotes an early divergence of b2 preproteins from the general matrix import pathway, precluding an unfolding role for mt-Hsp70 in the translocation of most of the mature portions of a preprotein. We propose a sorting model of cytochrome b2 which explains the apparently divergent previous results by a unifying hypothesis.     

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.     

Protein transport into mitochondria

Mitochondria are made up of two membrane systems that subdivide this organelle into two aqueous subcompartments: the matrix, which is enclosed by the inner membrane, and the intermembrane space, which is located between the inner and the outer membrane. Protein import into mitochondria is a complex reaction, as every protein has to be routed to its specific destination within the organelle. In the past few years, studies with mitochondria of Neurospora crassa and Saccharomyces cerevisiae have led to the identification of four distinct translocation machineries that are conserved among eukaryotes. These translocases, in a concerted fashion, mediate import and sorting of proteins into the mitochondrial subcompartments.     

Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.