Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).     

Nucleotide sequence of a Xenopus laevis mitochondrial DNA fragment containing the D-loop, flanking tRNA genes and the apocytochrome b gene

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial (mt) displacement (D) loop and surrounding genes [Wong et al., Nucl. Acids Res. 11 (1983) 4977-4995] are reported, including addition of two stretches of nucleotides and 60 scattered modifications. The additional sequences presented here correspond to the apocytochrome b gene, the tRNAGlu gene and part of URF6. This allows us to propose a conformational model for the X. laevis apocytochrome b protein and also permits comparisons with mammalian mtDNA. The D-loop sequence is poorly conserved except for sequences involved in the regulation of the mt genome (conserved sequence blocks and the DNA polymerase stop sequences). On the other hand, all genes show marked conservation both of their nucleotide sequence and their respective location on the mt genome. Organization of the genetic information described for mammalian mtDNA also holds for the X. laevis mtDNA. This result strongly suggests that all animal vertebrate mtDNAs have followed the same evolutionary pathway.     

The synthesis of cytochrome b on mitochondrial ribosomes in baker's yeast.

Antiserum against a major cytochrome b peptide isolated from yeast mitochondria as described previously (Lin, L.-F.H., and Beattie, D.S., J. Biol. Chem. 1978, 253, 2412--2418) was raised in rabbits and shown to be monospecific against the pure antigen. Mitochondria were isolated from yeast cells grown in [3H]leucine, extracted with Lubrol and treated with antiserum to cytochrome b. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of a single major band of molecular weight 31 000 corresponding to cytochrome b. In order to determine the intracellular site of translation of cytochrome b, yeast cells were labeled in vivo under non-growing conditions with [3H]leucine in the absence or presence of inhibitors of cytoplasmic and mitochondrial protein synthesis. The incorporation of radioactive leucine into the apoprotein of cytochrome b isolated by immunoprecipitation followed by gel electrophoresis was insensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and sensitive to acriflavin, erythromycin, and chloramphenicol (inhibitors of mitochondrial protein synthesis). Furthermore, no cytochrome b apoprotein was present in a cytoplasmic petite mutant which lacked mitochondrial protein synthesis. Cytochrome b is thus a product of protein synthesis on mitochondrial ribosomes.     

Limitations of allotopic expression of mitochondrial genes in mammalian cells

The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Cytochrome b, the var 1 protein, and subunits I and III of cytochrome c oxidase are synthesized without transient presequences in Saccharomyces cerevisiae

The N-termini of four mitochondrial translation products, the var 1 protein, cytochrome b, and subunits I and III of cytochrome c oxidase have been characterized in Saccharomyces cerevisiae and compared with the known DNA sequences of the respective structural genes. The four mature proteins correspond to the predicted primary translation products and retain the formylated methionine residue. Thus, subunit II of cytochrome c oxidase studied previously [Pratje et al. (1983) EMBO J.2, 1049-1054] is so far the only mitochondrial translation product carrying a N-terminal-extended transient presequence in S. cerevisiae.     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^? parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.     

Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562

The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.     

Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function

Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) $\text{Arch. Biochem. Biophys.}$ 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [³⁵S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme.     

Identification of the subunits of bovine NADH dehydrogenase which are encoded by the mitochondrial genome.

Products of the mitochondrial genome were identified in the bovine kidney cell line NBL-1 by labelling with [35S]methionine in the presence of cycloheximide. Seven proteins were precipitated by an antiserum to bovine heart NADH dehydrogenase, corresponding to the seven mitochondrial gene products identified in the human HeLa cell line. Comparison of these mitochondrial gene products with purified bovine NADH dehydrogenase by SDS/gel electrophoresis revealed that the ND-5 product is probably a previously unidentified protein of apparent Mr 51,000, and the ND-4 product is the protein of apparent Mr 39,000.