Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.     

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.     

Cytochrome b is necessary for the effective processing of core protein I and the iron-sulfur protein of complex III in the mitochondria

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in a mutant of yeast (W-267, Box 6-2) that lacks a spectrally detectable cytochrome b and synthesizes a shortened form of apocytochrome b. We recently reported that several cytochrome b-deficient mutants contained significantly diminished amounts of core proteins I and II as well as the iron-sulfur protein, but contained equal amounts of cytochrome c1 compared to the wild type (K. Sen and D. S. Beattie, Arch. Biochem. Biophys. 242, 393-401, 1985). In the present study, the time course of processing of precursors of both core protein I and the iron-sulfur protein which had accumulated in cells treated with the uncoupler carbonyl m-chlorophenyl hydrazone (CCCP) was noted to be significantly lower in the mutant compared to the wild type. The amounts of the mature forms of these proteins in mitochondria pulse labeled under different conditions was also considerably decreased at all times studied. The synthesis of both proteins appeared to be unaffected in the mutant, as the precursor forms of both proteins accumulated to the same extent when processing in vivo was blocked by CCCP. Furthermore, translation of RNA in a reticulocyte lysate in vitro indicated that the messenger RNAs for both proteins were present in the mutant and translated with equal efficiency. The import into isolated mitochondria of the precursor forms of the iron-sulfur protein synthesized in the cell-free system was also decreased in the mutant mitochondria. In addition, the precursor form was bound to the exterior of the mitochondrial membrane where it was sensitive to digestion with proteases. By contrast, the synthesis and processing of cytochrome c1 appeared to be unaffected in these mutants. These results suggest that cytochrome b is necessary for the proper processing and assembly of both core protein I and the iron-sulfur protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane.     

Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.     

Processing of the intermediate form of the iron-sulfur protein of the BC1 complex to the mature form after import into yeast mitochondria.

The Rieske iron-sulfur protein of the cytochrome bc1 complex is synthesized in the cytosol as a precursor with an additional 30 amino acids at the amino terminus. After import into the mitochondrial matrix, the precursor is processed to the mature form by two distinct proteolytic cleavages. Addition of 2.5 mM EDTA and 0.5 mM o-phenanthroline to the incubation mixture during import of the iron-sulfur protein precursor in vitro resulted in the selective inhibition of the second processing step with the concomitant accumulation of the intermediate form. The intermediate form was chased to the mature form in the presence of antimycin and oligomycin (to block the formation of a membrane potential) provided that 0.5 nM ATP and a metal ion such as Ca2+, Mn2+, or Mg2+ were added. Ca2+ ion was the most effective and at a concentration of 2.5 mM resulted in the complete cleavage of the intermediate to the mature form. Addition of Zn2+, Co2+, Mo2+, and Fe2+ was not effective in restoring the second cleavage. The pH optimum for the processing of the intermediate form of the iron-sulfur protein to the mature form was between 6.8-8.0. Processing of the intermediate form of the iron-sulfur protein to the mature form was observed at temperatures ranging from 12 to 27 degrees C in a temperature-dependent manner. The time course during the chase indicated that the second processing step was completed within 2 min after addition of Ca2+ ions. Attempts to isolate the second processing enzyme by sonication of mitochondria or by solubilization with detergents such as digitonin, Triton X-100, dodecyl-maltoside, or octyl-glucoside were unsuccessful as only the first cleavage was observed. Hence, the second processing enzyme may be present in the inner membrane or matrix in a conformation disrupted by detergents or alternatively the enzyme may be very labile.     

Kinetics of assembly of complex III into the yeast mitochondrial membrane. Evidence for a precursor to the iron-sulfur protein

Complex III immunoprecipitated from yeast cells labeled in vivo with [35S]sulfate or [3H]leucine contained seven subunits with molecular weights ranging from 15,000 to 47,000 when analyzed by electrophoresis on polyacrylamide gels. The subunit composition of the immunoprecipitates was identical with that of the purified complex III isolated from bakers' yeast suggesting that the antiserum recognizes the holoenzyme assembled properly in the membrane (Sidhu, A., and Beattie, D.S. (1982) J. Biol. Chem. 257, 7879-7886). Kinetic studies using double-labeled yeast cells followed by immunoprecipitation of complex III indicated that the subunits of the complex are assembled into the holoenzyme at very different rates. Cytochromes b and c1 and the 15,000-dalton subunit were the first polypeptides to be assembled into the complex with a half-time of labeling of 2.0-2.4 min. Core protein I and the iron-sulfur protein were inserted more slowly into the complex with a half-time of labeling of 4.6 and 5.3 min, respectively. Calculations of precursor pool sizes of the subunits indicated that for both core protein I and the iron-sulfur protein, there are large pools of precursors. The iron-sulfur protein was synthesized in vivo as a larger precursor polypeptide of molecular mass 28,000 Da. The precursor was subsequently cleaved, in a process requiring an energized mitochondrial inner membrane, into an intermediate form 1,500 Da larger than the mature subunit. The conversion of the intermediate to the mature form occurred in the inner mitochondrial membrane.     

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.     

Rhodamine 6G inhibits the matrix-catalyzed processing of precursors of rat-liver mitochondrial proteins

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the alpha and beta subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B.D. (1984) Eur. J. Biochem. 144, 387-392].     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

Immunological and chemical characterization of rat liver cytochrome c oxidase

Rat liver cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 different polypeptide chains. Specific antisera against the holoenzyme and against purified subunits IV and VIII were used to characterize the enzyme complex. The antiserum against subunit IV precipitates from sodium dodecyl sulfate-dissociated mitochondria only subunit IV and from Triton X-100-dissolved mitochondria all 12 polypeptide chains, indicating their integral location within the enzyme complex. Different antisera against the holoenzyme only precipitate subunits IV, V and VIb from sodium dodecyl sulfate-dissociated mitochondria, suggesting the location of these subunits on the surface layer of the complex. Subunit VIII is thought to be located within the complex, since a specific antiserum does not precipitate the complex. The amino acid composition of all 12 protein subunits is different, thus excluding their origin from proteolytic degradation. The proteolytic degradation of subunit IV into IV during isolation of the enzyme was corroborated by the very similar amino acid composition of both proteins.     

Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex

The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing.     

Cloning and sequence analysis of a cDNA encoding the Rieske iron-sulfur protein of rat mitochondrial cytochrome bc1 complex

We have isolated a cDNA clone for the Rieske iron-sulfur protein of rat cytochrome bc1 complex, by screening a rat liver cDNA expression library using antiserum directed against the corresponding protein of bovine. The amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that the mature polypeptide of the rat protein consists of 196 amino acid residues with a molecular weight of 21,465, and that it is formed as a precursor with an amino-terminal extension. Northern blot analysis indicated that rat liver possibly contains different sizes of mRNAs for the Rieske iron-sulfur protein, and Southern blot analysis demonstrated that rats and mice possess a single gene for this protein.     

Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

Biogenesis of the cytochrome bc1 complex of yeast mitochondria. A precursor form of the cytoplasmically made subunit V.

The cytoplasmically made subunit V of the yeast mitochondrial cytochrome bc1 complex is synthesized as a larger polypeptide in vitro. This was shown by programming a reticulocyte lysate with yeast RNA and immunoprecipitating the labeled translation products with a subunit V-specific antiserum. The larger form of subunit V could also be detected in pulse-labeled spheroplasts; upon a subsequent chase, most of it disappeared. A proteolytic fingerprint of the larger form was closely similar to that of the mature subunit. These data suggest that the cytoplasmically made subunit V is translated as a larger precursor which is cleaved to the mature subunit either during or after its entry into the mitochondria.     

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.     

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.