Import of cytochrome c into mitochondria. Cytochrome c heme lyase

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.     

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.     

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.     

Import of apocytochrome c into the mitochondrial intermembrane space along a cytochrome c1 sorting pathway

The question of whether cytochrome c could be functionally sorted to the mitochondrial intermembrane space along a "conservative sorting" pathway was investigated using a fusion protein termed pLc1-c. pLc1-c contains 3-fold targeting information, namely, the complete bipartite presequence of the cytochrome c1 precursor joined to the amino terminus of apocytochrome c. pLc1-c could be selectively imported into the intermembrane space either directly across the outer membrane along a cytochrome c import route or along a cytochrome c1 route via the matrix. Thus, apocytochrome c could be sorted along a conservative sorting pathway; however, following reexport from the matrix, apo-Lc1-c could not be converted to its holo counterpart. Despite the apparent similarity of structure and functional location of the heme lyases and similarity of the heme binding regions in their respective apoproteins, cytochrome c heme lyase and cytochrome c1 heme lyase apparently have different and nonoverlapping substrate specificities.     

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.     

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.     

Apocytochrome c: an exceptional mitochondrial precursor protein using an exceptional import pathway

The cytochrome c import pathway differs markedly from the general route taken by the majority of other imported proteins, which is characterized by the import involvement of namely, surface receptors, the general insertion protein (GIP), contact sites and by the requirement of a membrane potential (delta psi). Unique features of both the cytochrome c precursor (apocytochrome c) and of the mechanism that transports it into mitochondria, have contributed to the evolution of a distinct import pathway that is not shared by any other mitochondrial protein analysed thus far. The cytochrome c pathway is particularly unique because i) apocytochrome c appears to have spontaneous membrane insertion-activity; ii) cytochrome c heme lyase seems to act as a specific binding site in lieu of a surface receptor and; iii) covalent heme addition and the associated refolding of the polypeptide appears to provide the free energy for the translocation of the cytochrome c polypeptide across the outer mitochondrial membrane.     

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.     

A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria.     

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.     

Trypanosoma brucei cytochrome c1 is imported into mitochondria along an unusual pathway

In most eukaryotic organisms, cytochrome c(1) is encoded in the nucleus, translated on cytosolic ribosomes, and directed to its final destination in the mitochondrial inner membrane by a bipartite, cleaved, amino-terminal presequence. However, in the kinetoplastids and euglenoids, the cytochrome c(1) protein has been shown to lack a cleaved presequence; a single methionine is removed from the amino terminus upon maturation, and the sequence upstream of the heme-binding site is generally shorter than that of the other eukaryotic homologs. We have used a newly developed mitochondrial protein import assay system from Trypanosoma brucei to demonstrate that the T. brucei cytochrome c(1) protein is imported along a non-conservative pathway similar to that described for the inner membrane carrier proteins of other organisms. This pathway requires external ATP and an external protein receptor but is not absolutely dependent on a membrane potential or on ATP hydrolysis in the mitochondrial matrix. We propose the cytochrome c(1) import in T. brucei is a two-step process first involving a membrane potential independent translocation across the outer mitochondrial membrane followed by heme attachment and a membrane potential-dependent insertion into the inner membrane.     

Mitochondrial localization of cytochrome c1 with specific antibodies]

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.     

Effect of enzymatic methylation of apocytochrome c on holocytochrome c formation and proteolysis

1. Methylation of the lysine at residue 72 of yeast apocytochrome c increases its import into mitochondria. 2. Using methylated and unmethylated apocytochrome c as substrate and intact yeast mitochondria and a solubilized mitochondrial fraction as a source of cytochrome c heme lyase, the results show that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein. 3. The above result suggests that the import mechanism is separate from the heme-attaching activity. 4. Unmethylated apocytochrome c was less resistant to a yeast homogenate fraction that methylated apocytochrome c, suggesting that methylation of apocytochrome c alters the conformation of the whole protein.     

蛋白质跨线粒体膜的运送

线粒体约含１０００种左右蛋白质,其中９８％以上系由细胞核编码,在细胞质核酸体上以前体形式合成之后再运至线粒体并选分定位于各部分,现对定位于基持和内膜的蛋白质的运送途径研究的新进展作一扼要介绍,脱血红素细胞色素ｃ是细胞色素ｃ的前体,它既不含导肽,在线粒体外膜迄今也未发现共受体,对其转运的研究概况也作了评述。     

Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases

Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity. However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity. Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e. CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL. Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL. Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor. Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction.     

A bacterial cytochrome c heme lyase. CcmF forms a complex with the heme chaperone CcmE and CcmH but not with apocytochrome c.

Biogenesis of c-type cytochromes in Escherichia coli involves a number of membrane proteins (CcmA-H), which are required for the transfer of heme to the periplasmically located apocytochrome c. The pathway includes (i) covalent, transient binding of heme to the periplasmic domain of the heme chaperone CcmE; (ii) the subsequent release of heme; and (iii) transfer and covalent attachment of heme to apocytochrome c. Here, we report that CcmF is a key player in the late steps of cytochrome c maturation. We demonstrate that the conserved histidines His-173, His-261, His-303, and His-491 and the tryptophan-rich signature motif of the CcmF protein family are functionally required. Co-immunoprecipitation experiments revealed that CcmF interacts directly with the heme donor CcmE and with CcmH but not with apocytochrome c. We propose that CcmFH forms a bacterial heme lyase complex for the transfer of heme from CcmE to apocytochrome c.     

Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase.

Cytochrome c1, a subunit of the mitochondrial ubiquinol--cytochrome-c reductase, is synthesized on cytosolic ribosomes as a precursor protein of 37 kDa. Maturation to the mature 31-kDa form involves two proteolytic processing steps of the amino-terminal presequence. After removal of the amino-terminal part by the matrix-localized processing peptidase, the carboxy-terminal part of the presequence is cleaved off by an unknown intermembrane space protease. This step depends on covalent linkage of heme to the apoprotein. At least two complementation groups (I and II) can be distinguished among mutants of the yeast Saccharomyces cerevisiae, which are defective in this second proteolytic processing, i.e. they accumulate the intermediate-sized form of cytochrome c1 instead of the mature form. Recently, it was shown that complementation group II defines the structural gene for cytochrome c1 [Sadler, I., Suda, K., Schatz, G., Kaudewitz, F. & Haid, A., (1984) EMBO J. 3, 2137-2143]. We report on the molecular cloning and characterization of the CYT2 gene representing complementation group I. It maps on chromosome XI and encodes a mitochondrial protein of about 26 kDa. Extensive similarity to Neurospora crassa and S. cerevisiae cytochrome-c--heme lyase, as well as the phenotype of cyt2 mutants, strongly suggest that we have identified the gene for cytochrome-c1--heme lyase.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.     

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.     

Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.