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.     

Effect of enzymatic methylation of yeast iso-1-cytochrome c on its isoelectric point

Yeast iso-1- unmethylated and methylated apocytochrome c were synthesized in vitro by translating yeast cytochrome c mRNA, and by subsequently methylating the protein product. Unmethylated and methylated iso-1-holocytochrome c were extracted from Saccharomyces cerevisiae. By employing a column isoelectrofocusing technique, the pI values of these proteins were determined. The pI values of unmethylated and methylated apocytochrome c were found to be 9.60 and 8.70, respectively, with a difference of 0.90 pI unit. On the other hand, the pI values of unmethylated and methylated holocytochrome c were 9.72 and 9.68, respectively, with a difference of 0.04 unit. Therefore, although the pI values of both apo- and holocytochrome c decreased by methylation, methylation of apocytochrome c had a more profound effect on the pI of the protein. The result also indicated that conjugation of heme to apocytochrome c increased its pI value, resulting in the more "compact" and basic structure of the protein. The observed magnitude of the pI change subsequent to the methylation of apocytochrome c (decrease of 0.90 unit) seemed to be contradictory to the predicted increase in the value, since the positive charge is fixed on the quaternary amino group of trimethyllysine and there is no proton to titrate. Trimethylation of epsilon-NH2 group of Res-72 lysine of apocytochrome c could disrupt any possible hydrogen bond formed by the nitrogen atom of Res-72 lysine residues, as visualized by a space-filling model. The model and observed shift in the "effective charge" of the protein strongly suggest that conformational change in the apoprotein takes place upon methylation. This presumably altered conformation along with the decrease in pI caused by methylation may play a role in enhancement of apocytochrome c import into mitochondria.     

Cytochrome c methyltransferase, Ctm1p, of yeast

Cytochromes c from plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. Testing of 6,144 strains of S. cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c. We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c. Unmethylated forms of cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S. cerevisiae or Escherichia coli. Iso-1-cytochrome c purified from a ctm1-Delta strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro. Like apocytochrome c, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c. The ctm1-Delta strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.     

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.     

Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria.

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.     

Loading rat liver mitochondria with apocytochrome c provokes exit of cytochrome c

Rat liver mitochondria were loaded with cytochrome c by incubation with large amounts of [14C]apocytochrome c. After being washed they were incubated with either more apocytochrome c or cytochrome c. There was no release of labeled proteins from the mitochondria when incubated with cytochrome c. However, there was when incubated with apocytochrome c. The material released showed only one radioactive band which migrated as cytochrome c. Also no release of proteins other than cytochrome c was detected when liver mitochondria isolated from rats injected with [35S]methionine were incubated with apocytochrome c. These results suggest that the level and possibly the turnover of cytochrome c in rat liver mitochondria is regulated by the entry of apocytochrome c into mitochondria.     

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.     

In vivo studies on yeast cytochrome c methylation in relation to protein synthesis

Methylation of cytochrome c was studied in vivo using double label with L-[methyl-3H]methionine and DL-[2-14C]methionine. In pulse-chase experiments the cytochrome c associated with the mitochondrial fraction possessed a higher ratio of 3H/14C label, suggesting the presence of methylated cytochrome c. The appearance of methylated cytochrome c in mitochondria showed no lag phase. The inhibition of cytochrome c methylation in presence of cycloheximide indicated that both the methylation and protein synthesis were tightly coupled and cycloheximide selectively inhibited cytochrome c methylation. There was also an indication of selective turnover of incorporation methyl groups in preformed cytochrome c.     

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.     

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.     

Effect of methylation on the stability of cytochrome c of Saccharomyces cerevisiae in vivo

The in vivo stability of methylated and unmethylated cytochrome c in Saccharomyces cerevisiae was studied by pulse-labeling the hemoproteins with [methyl-3H]-methionine and/or [2-14C]methionine and following the fate of these proteins under anaerobiosis and in the presence of cycloleucine. These two conditions will respectively block further cytochrome c synthesis and inhibit methylation by lowering the cellular S-adenosyl-L-methionine pool and, thus, permit an unambiguous interpretation of the data. The results showed that the rate of degradation of unmethylated cytochrome c was constant throughout the chase period, while methylated cytochrome c degradation was seen only in the later part of cold chase. At the end of the chase period (40 h), the extent of degradation of the unmethylated species was three times higher than the methylated species. This indicated that the methylation of cytochrome c has a protective effects against the intracellular proteolytic enzyme attack on itself. Furthermore, this protective effect was considerably reduced in the petite mutant, which lacks high affinity cytochrome c binding sites, functional cytochrome c reductase, and oxidase, and possesses a less integrated and organized mitochondrial membrane. These results led us to the conclusion that the mechanism of methylated cytochrome c stabilization is best explained by a higher efficacy of binding to the mitochondria.     

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.     

Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.

Molecular cloning and characterization of cytochrome c cDNA clones of Neurospora crassa wild-type (74A) and a cytochrome c-deficient mutant (cyc1-1) are described. Southern blot analysis of genomic DNA indicates that only one cytochrome c gene exists in the N. crassa genome. The cDNA sequence of the wild-type cytochrome c confirmed the previously determined protein sequence. Sequence analysis of the cyc1-1 cDNA for cytochrome c revealed the presence of a larger open reading frame, owing to the presence of an unspliced intron in the 3' end of the coding region. Splicing of this intron is obviously prevented due to the presence of two base exchanges in the highly conserved intron consensus sequences. Consequently, cyc1-1 synthesizes apocytochrome c with an altered carboxy terminus, 19 amino acids longer than the wild-type cytochrome c, with the final 27 amino acids being of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and, consequently, import into mitochondria. Thus, unlike other mitochondrial precursor proteins, where it has been demonstrated that the amino terminus alone is sufficient to target the protein to the mitochondria, an intact carboxy terminus is required for efficient import of apocytochrome c into mitochondria. This is independent confirmation for the view that the import pathway of cytochrome c is unique with respect to all other mitochondrial proteins studied to date.     

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.     

Mmicular mechanisms of cytochrome c biogenesis: three distinct systems

The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c -type cytochromes. The hallmark of c -type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys–Xxx–Yyy–Cys–His signature motif). From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein. In this review, common principles of assembly for all systems and the mmicular mechanisms predicted for each system are summarized. Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction. Accessory proteins of systems I and II co-ordinate the positioning of these two substrates at the membrane surface for covalent ligation. The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates. For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.     

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.     

A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.     

脱辅基细胞色素c定向于线粒体的转运特异性研究

用分离纯化的完整线粒体和部分细胞器组分,初步研究了脱辅基细胞色素c在细胞内转运的特异性。完整线粒体用差速离心和密度梯度离心的方法,从幼龄鸡心肌组织中获得,对胞内几种细胞器标志酶比活力的测量表明,纯化的线粒体单胺氧化酶活力提高25.6倍,腺苷酸激酶活力提高3.59倍,细胞色素c氧化酶活力提高5.48倍,外膜完整性达90%以上,呼吸控制率大于20。以上数据表明该纯化的线粒体受胞内其它囊泡成分污染少,外膜完整并具有较高的氧化磷酸化偶联效率;在纯化线粒体的同时,得到另两种细胞器组分-内质网和溶酶体囊泡。体外转录翻译的apo.c与上述几个组分的结合实验表明,完整线粒体与apo.c的结合能力明显高于其它组分。