Rieske Iron-sulfur Protein Research Articles

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the α and β subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75-5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.

The thylakoid-targeting domain of the chloroplast Rieske iron-sulfur protein is located in the N-terminal hydrophobic region of the mature protein

The thylakoid-targeting domain of the Rieske FeS protein has been located in the N-terminal 55 amino acids of the mature protein by importing chimeric and truncated precursor proteins into isolated pea chloroplasts. A chimeric protein consisting of the presequence of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fused to the mature Rieske protein sequence was imported by chloroplasts, processed in the stroma, and translocated into the thylakoids, indicating that the thylakoid-targeting information was located within the mature Rieske protein. A truncated Rieske protein precursor consisting of the presequence and the N-terminal 55 amino acids of the mature protein was imported by chloroplasts and routed to the thylakoids, indicating that the thylakoid-targeting domain of the Rieske protein is located within the predominantly hydrophobic N-terminal region of the mature protein. Chimeric proteins consisting of this truncated Rieske protein precursor fused to the mature Rubisco small subunit and to plastocyanin were efficiently imported by chloroplasts and translocated across the thylakoid membrane. A proton-motive force was necessary for thylakoid translocation of the mature Rieske protein, the truncated Rieske protein, and the fusion protein consisting of the truncated Rieske protein and the mature Rubisco small subunit. In contrast, plastocyanin and the fusion of the truncated Rieske protein with plastocyanin were translocated across the thylakoid membrane in the presence of nigericin and valinomycin, indicating that the energy requirement for thylakoid translocation was conferred by the passenger protein and not by the thylakoid-targeting sequence.

Isolation and characterization of QCR10, the nuclear gene encoding the 8.5-kDa subunit 10 of the Saccharomyces cerevisiae cytochrome bc1 complex.

We have cloned and sequenced QCR10, the nuclear gene encoding an 8.5-kDa protein proposed to be a subunit of the cytochrome bc1 complex of Saccharomyces cerevisiae. QCR10 includes a 231-base pair open reading frame capable of encoding a protein of 77 amino acids with a predicted molecular mass of 8492 Da. The codons for the amino-terminal methionine and alanine are separated from the remainder of the open reading frame by a 63-base pair intron that contains 5'-donor, 3'-acceptor, and TACTAAC sequences known to be necessary for splicing. The deduced amino acid sequence of the 8.5-kDa yeast protein is 28% identical to that of the 6.4-kDa subunit 11 from the bovine heart cytochrome bc1 complex, and the predicted secondary structures of the two proteins are very similar. Deletion of the chromosomal copy of QCR10 did not affect the growth of yeast on nonfermentable carbon sources. However, deletion of QCR10 synergistically contributes to the temperature-dependent phenotype resulting from deletion of QCR6, the gene for subunit 6 of the cytochrome bc1 complex. In addition, ubiquinol-cytochrome c oxidoreductase activity was reduced 40% in mitochondrial membranes from the QCR10 deletion strain, and the Rieske iron-sulfur protein was lost when the cytochrome bc1 complex lacking the 8.5-kDa protein was purified. We conclude that the 8.5-kDa protein encoded by QCR10 is a subunit of the yeast cytochrome bc1 complex and that the presence of this subunit during assembly is required for stable association of the iron-sulfur protein with the complex.

Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein.

The mode of membrane attachment of five subunits from Chlamydomonas reinhardtii cytochrome b6f complex has been studied using biochemical approaches. Antisera specific for cytochrome f, cytochrome b6, the Rieske iron-sulfur protein, subunit IV, and a 4-kDa subunit (product of the petG gene) were used to quantify the degree of extraction of each of these polypeptides following various treatments. In contrast to the other four subunits, the Rieske protein was extracted to extents varying between 50 and 100% following two cycles of freezing and thawing in the presence of chaotropic agents (KSCN, urea, or NaI). The Rieske protein was not extracted by 2 M NaCl and was rather resistant to alkaline treatments, being extracted by 20 mM 3-(cyclohexylamino)propanesulfonic acid buffer only at pH > 11.5. The hydrodynamic behavior of the isolated Rieske protein was examined in the absence and presence of detergent by ultracentrifugation and by molecular sieving. The extracted protein bound neither to laurylmaltoside nor to C12E8 micelles. Its sedimentation coefficient (D20,w = 9.6 x 10(-11) m2 x s-1), diffusion coefficient (s20,w = 2S), an deduced molecular mass (20.0 +/- 1.7 kDa) are those expected for the monomeric protein. We conclude that the Rieske protein is extrinsic and therefore does not cross the membrane, although its association with the rest of the complex involves primarily hydrophobic interactions, and that the other four subunits analyzed are intrinsic.

Characterization of the gene of the chloroplast Rieske iron-sulfur protein in Chlamydomonas reinhardtii. Indications for an uncleaved lumen targeting sequence.

The sequence of the nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome b6f complex of Chlamydomonas reinhardtii has been established. Comparison of genomic clones and amplified cDNA indicates that the petC gene is interrupted by four introns within the coding sequence of the mature protein. The nucleotide sequence predicts a precursor protein of 206 amino acid residues with a transit peptide of 29 amino acids. The transit peptide is shorter than that of higher plants and has a basic region typical for the transfer through the chloroplast envelope, but no hydrophobic segment at the C-terminal end as is found in proteins transferred through the thylakoid membrane. The mature protein shows a high degree of homology with that of higher plants and has an N-terminal hydrophobic segment as in other Rieske proteins. Biochemical data (Breyton, C., de Vitry, C., and Popot, J.-L. (1994) J. Biol. Chem. 269, 7597-7602) indicate that the chloroplast Rieske protein of C. reinhardtii is an extrinsic membrane protein. Therefore, this N-terminal hydrophobic segment is not a transmembrane segment but may act as an uncleaved N-terminal thylakoid membrane transfer signal sequence. There are other examples of uncleaved hydrophobic membrane transfer signal in secreted proteins, although these are rare.

Protease maturation of the Rieske iron-sulphur protein after its insertion into the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae

Protease maturation of the Rieske iron-sulphur protein after its insertion into the mitochondrial cytochrome <i>bc</i>1 complex of <i>Saccharomyces cerevisiae</i>

The cytochrome b 6f complex

The cytochrome b 6f complex in oxygenic photosynthetic membranes is believed to have many structure-function analogies to the cytochrome bc 1 complex found in the mitochondrial respiratory chain and in chromatophore membranes of purple photosynthetic bacteria. Three polypeptides in the complex, cytochrome f, cytochrome b 6 and the Rieske iron-sulfur protein contain redox prosthetic groups. Cytochromes f and b 6 are anchored to the membrane by one transmembrane helix and four transmembrane helices respectively. Recent studies on the Rieske high-potential iron-sulfur protein suggest that it may be an extrinsic membrane protein. As in mitochondria, the complex that is active in vitro is predominantly dimeric and it has not yet been possible to obtain a monomeric complex that has significant activity. The crystal structure of the major (252 residues, 87% of the protein) lumenal domain of cytochrome f has been solved to high (2.3 Å) resolution. The elongate predominantly β-strand structure, reminiscent of the motif of animal cell surface proteins, and the amino-terminal α-amino group acting as an axial heme ligand are unique features of the structure. This ligation provided an unexpected insight into the mechanism of translocation of the protein into the membrane. The structure makes specific predictions about the location of the positively charged docking site for the electron acceptor, plastocyanin.

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma.

The precursor of the Rieske FeS protein, a thylakoid membrane protein, was imported by isolated pea chloroplasts, and the mature protein was shown to be integrated into the cytochrome bf complex of the thylakoid membranes. Insertion into the thylakoid membrane was sensitive to the ionophores nigericin and valinomycin, suggesting a requirement for a proton motive force. A considerable proportion of the imported Rieske protein was detected in the stromal fraction of the chloroplasts, and this increased when membrane insertion was blocked with ionophores. Electrophoresis of the stromal fraction under nondenaturing conditions resolved two distinct complexes containing the Rieske protein. One of these complexes was identified as an association of the Rieske protein with the chaperonin Cpn60 complex by its electrophoretic mobility, Mg-ATP-dependent dissociation, and immunoprecipitation with anti-Cpn60 antibodies. Coimmunoprecipitation of imported Rieske protein with anti-heat shock protein 70 (Hsp70) antibodies indicated that the Rieske protein was also associated, in an ATP-dissociable form, with a chloroplast Hsp70 homolog. Immunoprecipitation analysis of an import time course detected the highest amounts of the Cpn60-Rieske protein complex early in the time course, whereas highest amounts of the Hsp70-Rieske protein complex were formed much later. The disappearance of the Cpn60-Rieske protein complex correlated with increased amounts of the Rieske protein in the thylakoid fraction.

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma

Assignment of the gene for the core protein II (UQCRC2) subunit of the mitochondrial cytochrome bc1 complex to human chromosome 16p12.

The mammalian cytochrome be[sub 1] complex (complex III) of the mitochondrial respiratory chain catalyzes electron transfer from ubiquinol to cytochrome c. The complex consists of 10-11 subunits: Core proteins I and II, cytochromes b and c[sub 1], the Rieske iron-sulfur protein, the ubiquinone-binding protein, the hinge protein, and 3-4 subunits of low molecular weight. Cytochrome b is encoded by the mitochondrial genome; the other subunits are encoded by nuclear genes. Both the human cytochrome c[sub 1] and the human ubiquinone-binding protein subunits have been assigned to chromosome 8 by somatic cell hybrid mapping. In this study, the authors used in situ hybridization to map core protein II. In situ hybridization to BrdU-synchronized peripheral blood lymphocytes was performed using the method of Harper and Saunders. Chromosomes were stained with a modified fluorescence, 0.25% Wright's stain procedure. The positions of silver grains directly over or touching well-banded metaphase chromosomes were mapped to an ISCN idiogram. The analysis of the distribution of 200 silver grams following in situ hybridization revealed a significant clustering of grains in the p12 region of chromosome 16. The assignment of the core II subunit to human chromosome 16p12 confirms that it is encoded by the nuclear, rather thanmore » the mitochondrial, genome. The identification of a single strong hybridization signal is indicative of one locus with no pseudogenes. 6 refs., 1 fig.« less

Subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein

Deletion of QCR9, the nuclear gene encoding the 7.3-kDa subunit 9 of the cytochrome bc1 complex, impairs respiration of Saccharomyces cerevisiae, coincident with loss of ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b. Synthesis of cytochrome b by mitochondria from the deletion strain is unimpaired, indicating that the diminished b absorption is due to a post-assembly effect on the heme environment resulting from the absence of subunit 9. Iron-sulfur protein is present in normal amounts and processed to its mature form in the absence of subunit 9, although the protein is more labile to endogenous proteases during the isolation of membranes. EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present. Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in the subunit 9 deletion strain, which suggests that an EPR-silent, sluggishly reactive derivative of the iron-sulfur cluster is present. These results suggest that in the absence of subunit 9 the conformation of iron-sulfur protein is altered such that the protein is more labile, the iron-sulfur cluster is not properly inserted, and iron-sulfur protein interaction with cytochrome b is modified in a manner which distorts the heme environment. This is the first instance in which deletion of one of the supernumerary subunits of the cytochrome bc1 complex results in the loss of function of a redox center within the complex, without a concomitant loss of other subunits.

Mitochondrial ubiquinol-cytochrome c reductase complex: crystallization and protein: ubiquinone interaction.

The ubiquinol-cytochrome c reductase complex was crystallized in a thin plate form, which diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex contains ten protein subunits and 140 nmol phospholipid per milligram protein. Over 90% of the phospholipid and ubiquinone in the reductase can be removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The delipidated complex has no enzymatic activity and shows significant changes in the circular dichroism spectrum in the near UV region and in the EPR characteristics of both cytochromes b. Enzyme activity and spectral characteristics can be restored by replenishing the phospholipid and ubiquinone. The structural requirements of ubiquinone for electron transport were studied by measuring the ability of a variety of synthetic ubiquinone derivatives to restore the enzymatic activity and native spectroscopic signatures to the delipidated complex. Q-binding proteins and binding domains were identified using photoaffinity labeled Q-derivatives and HPLC separation of photolabeled peptides. Interaction between ubiquinol-cytochrome c reductase and succinate-Q reductase was established by differential scanning calorimetry and saturation transfer EPR using spin-labeled ubiquinol-cytochrome c reductase. Involvement of iron-sulfur protein in proton translocation by ubiquinol-cytochrome c reductase was investigated by hematorporphyrin-promoted photoinactivation of the complex. The cDNAs encoding the Rieske iron-sulfur protein and a small molecular mass Q-binding protein (QPc-9.5 kDa) were isolated and their nucleotide sequences determined. These will be useful in future structural and mechanistic studies of ubiquinol-cytochrome c reductase via in vitro reconstitution between an over-expressed, mutated subunit and a specific subunit-depleted reductase.

The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.

The amino acid sequence of subunit 9 of the bovine heart cytochrome bc1 complex is identical to the 78-amino acid presequence that is removed post-translationally from the Rieske iron-sulfur protein as it is imported and targeted to the mitochondrial cytochrome bc1 complex. Iron-sulfur protein precursor, generated by in vitro transcription and translation, is processed to mature size in a single step when incubated with rat liver mitochondria, and generates a peptide that comigrates on SDS-polyacrylamide gel electrophoresis with subunit 9. These results suggest that the Rieske protein is processed in a single proteolytic step after it is inserted into the cytochrome bc1 complex in mammals, and that the processed presequence remains as a subunit of the complex. This is apparently the first instance in which a cleaved targeting presequence has been shown to be retained in the cell, possibly exhibiting a second function in addition to its function in protein trafficking.

Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae.

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobilis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome bc1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; Em = 116 mV), cytochrome c1 (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric alpha-band with a maximum at 564 nm and a shoulder at 559 nm. The alpha bands of cytochrome bH and cytochrome c1 peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained. Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced.(mol of cytochrome c1)-1.s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc1 complexes.

Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002.

The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.

Import of the precursor of the chloroplast Rieske iron-sulphur protein by pea chloroplasts.

cDNA clones encoding the precursor of the Rieske FeS protein of the chloroplast thylakoid membrane have been isolated from a pea leaf cDNA library in lambda gt 11, following screening with antibodies to purified pea chloroplast Rieske FeS protein. The longest cDNA insert of 880 bp encodes a polypeptide of 230 amino acid residues, of which 50 residues constitute an N-terminal cleavable presequence and 180 residues make up the mature protein. Transcription and translation of the cDNA in vitro produced a polypeptide of 26 kDa which was efficiently imported by isolated pea chloroplasts and processed to the mature 20 kDa protein. Southern hybridisation to pea genomic DNA indicated the presence of a single gene encoding the Rieske FeS protein in the haploid genome.

Processing of the intermediate form of the iron-sulfur protein of the bc 1 complex to the mature form after import into yeast mitochondria

The Rieske iron-sulfur protein of the cytochrome bc 1 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 m m EDTA and 0.5 m m 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 m m ATP and a metal ion such as Ca 2+, Mn 2+, or Mg 2+ were added. Ca 2+ ion was the most effective and at a concentration of 2.5 m m resulted in the complete cleavage of the intermediate to the mature form. Addition of Zn 2+, Co 2+, Mo 2+, and Fe 2+ 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 °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 Ca 2+ ions. Attempts to isolate the second processing enzyme by sonication of mitochondria or by solubilization with detergents such as digitonin, Triton X-100, dodecylmaltoside, 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.

Characterization of the pet operon of Rhodospirillum rubrum

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c 1 components of the cytochrome bc 1 complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc 1 complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.

Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants.

cDNA clones coding for the nuclear-encoded mitochondrial Rieske iron-sulfur protein (RISP) have been isolated from maize and tobacco. Complementation analysis of hybrid proteins consisting of different domains of plant and yeast RISPs showed that the carboxyl two-thirds of the plant protein is functionally equivalent to that of the yeast protein. The amino terminus of the RISP, however, seems to be species specific because this region is not interchangeable between plant and yeast proteins. Complementation analysis of hybrid proteins also identified a structurally conserved domain probably essential for the function of bc1 complex RISPs. A specific domain from the plant RISP was found to cause temperature-sensitive respiratory growth in yeast. We have demonstrated that yeast can serve as a model system to study the structural and functional relationships of plant gene products that are enzymatic components of the mitochondrial respiratory chain.