Nuclear genes for mitochondrial proteins. Identification and isolation of a structural gene for subunit V of yeast cytochrome c oxidase

The gene for yeast cytochrome c oxidase subunit V, COX5, has been isolated from a Saccharomyces cerevisiae DNA library by complementation of a cytochrome c oxidase subunit V mutant, JM28. One complementing plasmid, YEp13-511, with a DNA insert of 4.8 kilobase pairs, has been characterized in detail. This plasmid restores respiratory competency in JM28, results in increased cytochrome c oxidase activity and a new form of subunit V in JM28 mitochondria, and is capable of selecting mRNA for subunit V. These results indicate that YEp13-511 carries the COX5 gene and that the subunit V encoded by this plasmid gene is capable of entering the mitochondrion and assembling into a functional holocytochrome c oxidase.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Targeted deletion of a yeast enolase structural gene. Identification and isolation of yeast enolase isozymes

Yeast contain two nontandemly repeated enolase structural genes which have been isolated on bacterial plasmids designated peno46 and peno8 (Holland, M. J., Holland, J. P., Thill, G. P., and Jackson, K. A. (1981) J. Biol. Chem. 256, 1385-1395). In order to study the expression of the enolase genes in vivo, the resident enolase gene in a wild type yeast strain corresponding to the gene isolated on peno46 was replaced with a deletion, constructed in vitro, which lacks 90% of the enolase coding sequences. Three catalytically active enolases are resolved differ DEAE-Sephadex chromatography of wild type cellular extracts. As expected, a single form of enolase was resolved from extracts of the mutant cell. Immunological and electrophoretic analyses of the multiple forms of enolase confirm that two enolase genes are expressed in wild type cells and that isozymes are formed in the cell by random assortment of the two polypeptides into three active enolase dimers. The yeast enolase loci have been designated ENO1 and ENO2. The deletion mutant lacks the enolase 1 polypeptide confirming that this polypeptide is encoded by the gene isolated on peno46. The intracellular steady state concentrations of the two polypeptides are dependent on the carbon source used to propagate the cells. Log phase cells grown on glucose contain 20-fold more enolase 2 polypeptide than enolase 1 polypeptide, whereas cells grown on ethanol or glycerol plus lactate contain similar amounts of the two polypeptides. The 20-fold higher than in cells grown on the nonfermentable carbon sources. In vitro translation of total cellular RNA suggests that the steady state concentrations of the two enolase mRNAs in cells grown on different carbon sources are proportional to the steady state concentrations of the respective enolase polypeptides.     

Cocrystals of yeast cytochrome c peroxidase and horse heart cytochrome c

Yeast cytochrome c peroxidase and horse heart cytochrome c have been cocrystallized in a form suitable for x-ray diffraction studies and the structure determined at 3.3 A. The asymmetric unit contains a dimer of the peroxidase which was oriented and positioned in the unit cell using molecular replacement techniques. Similar attempts to locate the cytochrome c molecules were unsuccessful. The peroxidase dimer model was subjected to eight rounds of restrained parameters least squares refinement after which the crystallographic R factor was 0.27 at 3.3 A. Examination of a 2Fo-Fc electron density map showed large "empty" regions between peroxidase dimers with no indication of cytochrome c molecules. Electrophoretic analysis of the crystals demonstrated the presence of the peroxidase and cytochrome c in an approximate equal molar ratio. Therefore, while cytochrome c molecules are present in the unit cell they are orientationally disordered and occupy the space between peroxidase dimers.     

Identification of cytochrome c oxidase subunits in nuclear yeast mutants lacking the functional enzyme.

Yeast mutants specifically lacking cytochrome c oxidase activity were screened for cytochrome c oxidase subunits by one- and two-dimensional electrophoresis, electrophoresis in exponential gradient gels, and immunoprecipitation with antisera against one or more of the cytoplasmically made subunits of the enzyme. Two cytochrome c oxidase-less nuclear mutants previously described from this laboratory each lack one or more mitochondrially synthesized cytochrome c oxidase subunits while possessing all four cytoplasmically synthesized subunits of that enzyme. The subunits remaining in these mutants were not assembled with each other; the cytoplasmically made subunits IV and VI could be released from the mitochondria by sonic oscillation, in contrast to the situation in wild type cells. No electrophoretically detectable alterations were found in any of the cytochrome c oxidase subunits present in the mutants. Nuclear mutations may thus cause both a loss as well as a defective assembly of mitochondrially made cytochrome c oxidase subunits.     

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc–CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.     

Genetic and physical analysis of the mitochondrial gene for subunit II of yeast cytochrome c oxidase.

The mitochondrial genetic locus oxi 1 contains the structural gene for subunit II of Cytochrome c oxidase. In this study, the oxi 1 locus, or at least a major portion of it, has been localized to a 2·4 kb2 HpaII fragment of mitochondrial DNA, by examining the mtDNA of oxi 1 mutants, and rho^? yeast strains that selectively retained in amplified form, this region of the mitochondria) genome. The 2·4 kb fragment is missing from the mtDNA of an oxi 1 locus deletion mutant, but is present in the mtDNAs retained by two rho^? strains that genetically recombine with all 16 oxi 1 mutants tested, to produce respiring progeny. Two other rho^? strains, that retained different but overlapping portions of the oxi 1 locus as determined genetically, contained mtDNAs consisting of “cloned” segments derived from within the 2·4 kb fragment: these rho^? mtDNAs hybridized only to the 2·4 kb HpaII fragment of wild-type mtDNA and could not be cleaved with HpaII. Furthermore, these two rho^? mtDNAs were found to correspond to sequences from opposite sides of the 2·4 kb fragment that overlap for 100 to 300 base-pairs near the middle of the fragment. Thus, five oxi 1 mutations that recombine with both of these rho^? strains could be further localized to this relatively short region of overlap. One such mutation, of particular interest because it produces an altered form of subunit II, was shown to lie on a 75-base-pair fragment that maps in this region of the overlap. The 75-base-pair fragment from the mutant migrates slightly faster during electrophoresis than the corresponding wild-type fragment. In contrast, the mobility of the fragment from a spontaneous revertant was indistinguishable from wild type.     

Kinetics of glucose repression of yeast cytochrome c.

The kinetics of glucose repression of cytochrome c synthesis was measured by a radioimmune assay. When 5 or 10% glucose was added to a derepressed culture, the rate of cytochrome c synthesis was reduced to the repressed level with a half-life of 2 min. The addition of 1 or 0.5% glucose repressed the rate of cytochrome c synthesis to the same level as high glucose concentrations but with a longer half-life of 3 min. Glucose repression had no effect on the stability or function of the cytochrome c protein. Cellular levels of active cytochrome c mRNA during glucose repression were measured by translation of total cellular polyadenylic acid-containing RNA and immunoprecipitation cytochrome c from the translation products. The results of these measurements indicate that glucose represses the rate of cytochrome c synthesis through a reduction in the level of translatable cytochrome c mRNA.     

Preparation of cytochrome c peroxidase from baker's yeast.

A simple and readily reproducible procedure is presented for the preparation and purification of cytochrome c peroxidase from baker's yeast. Following autolysis of the yeast and extraction, the enzyme is collected on DEAE-cellulose at moderately high ionic strength, cluted, concentrated, and subjected to gel filtration in 0.1 m sodium acetate buffer, pH 5.0. The properties of the crude preparation make gel filtration in this buffer suitable for near-final purification of the heme protein. The enzyme is then easily crystallized by dialysis.     

Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes.

Mouse contains two functional, but differentially expressed, cytochrome c genes. One of these genes is expressed in all somatic tissues so far examined. The other gene is expressed only in testis and is assumed to be spermatogenesis-specific. The nucleotide sequence of four mouse cytochrome c-like genes has been determined. One of these genes (MC1) contains an intron and encodes a polypeptide sequence identical to the published mouse somatic cytochrome c amino acid sequence. The other three genes can not properly encode a mouse cytochrome c protein and appear to be pseudogenes which have arisen via an insertion into the mouse genome of a cDNA copy of a cytochrome c mRNA molecule.     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

Expression of modified cytochrome c1 genes and restoration of the respiratory function in a yeast mutant lacking the nuclear cytochrome c1 gene

Yeast cytochrome c1 is a component of complex III, an oligomeric enzyme of the mitochondrial respiratory chain. In order to investigate the structural requirement of cytochrome c1 for the function and assembly of the enzyme, we used an in vivo complementation assay to determine whether or not an in vitro mutated cytochrome c1 is functional. A yeast mutant whose nuclear cytochrome c1 gene was specifically inactivated was constructed by means of a gene disruption technique. The mutant was unable to respire, and lacked spectrally and immunochemically detectable cytochrome c1. These defects disappeared on the introduction of a plasmid carrying the cytochrome c1 gene coding the wild-type molecule or one coding a mutant molecule lacking the carboxyl (C)-terminal 17 amino acid residues. On the other hand, another mutant gene with a deletion corresponding to the C-terminal 71 residues showed no such ability. These results suggest that the region between the C-terminal 17 and 71 residues is necessary for the function of cytochrome c1.     

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.     

Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.