Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.     

Phe356 in the yeast Ca2+ channel component Mid1 is a key residue for viability after exposure to alpha-factor

The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe356. After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe356 and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe356 caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe356 caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe356 residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance.     

Isolation and characterization of two alleles of the chicken cytochrome c gene.

Analysis of total chicken DNA by genomic blot hybridization indicates that only one cytochrome c gene exists in the chicken genome. The two alleles of this single cytochrome c gene have been isolated from a Charon 4A-chicken genomic library. This isolation made use of the yeast CYC1 cytochrome c gene as a specific hybridization probe. The 2 chicken alleles, CC9 and CC10, have been sequenced. The amino acid sequence predicted by these 2 alleles is identical, and agrees with the published chicken cytochrome c protein sequence. The flanking regions of these 2 alleles exhibit approximately 1% divergence, indicating a very limited polymorphism. Comparative sequence analysis with the flanking regions of previously isolated cytochrome c genes (yeast and rat) indicate no significant regions of homology. The presence of only one cytochrome c-like sequence in the chicken genome is in striking contrast with mammalian genomes, which contain as many as 20-30 cytochrome c-like sequences.     

Yeast cytochrome b2 gene: isolation with antibody probes

An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100). This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins. Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2. by DNA sequence analysis. One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence.     

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.     

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.     

Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector.

A phagemid (pING4) carrying the yeast iso-1-cytochrome c gene was constructed which bears all the elements necessary for replication in yeast and bacteria and may be converted into a single-stranded form of DNA for site-directed mutagenesis and nucleotide sequencing. The recombinant vector was used to create a complete set of 19 amino acid changes at position 82, a phylogenetically conserved phenylalanine residue in mitochondrial cytochrome c. All the different forms of cytochrome c were functional in vivo, based upon their ability to support respiration when the mutant proteins were expressed in a yeast strain (otherwise devoid of cytochrome c) grown on non-fermentable carbon sources, with only the strain containing the Cys82 variant having a substantially decreased growth rate. These results are interpreted in terms of the available structural and functional information previously reported on a subset of cytochrome c proteins with mutations at position 82.     

An in silico analysis of cytochrome c from Phanerochaete chrysosporium: its amino acid sequence and characterization of gene structural elements

An in silico approach was used to investigate cytochrome c and the cytochrome c gene of Phanerochaete chrysosporium. The cytochrome c gene contains four introns. Omission of the introns reveals a DNA sequence coding for a complete predicted amino acid sequence for P. chrysosporium cytochrome c consistent with those of other cytochromes c. Fungal cytochromes c often have a short N-terminal peptide preceding a Gly that is the N-terminal amino acid in many cytochromes c. Thus a microexon codes for an N-terminal pentapeptide (MetProTyrAlaPro) in P. chrysosporium that is identical to the N-terminal pentapeptide of Schizosaccharomyces pombe, a well studied yeast, the genome of which bears more similarity to higher eukaryotes than to other fungi. The fourth intron, when omitted, reveals the presence of another microexon resulting in a sequence for the C-terminal portion of the protein and the stop codon. Interestingly, two interpretations for the sequence of this intron leads to predictions that the C-terminal sequence ends with either AlaValAsn or AlaTyr. Selected aspects of the molecular architecture of cytochrome c and regulatory control elements of the P. chrysosporium cytochrome c gene were analyzed and compared to those present in other fungi and to those present in genes for lignin peroxidases and cytochromes P-450, two important families of hemeproteins produced by this fungus.     

Nucleotide sequence and evolution of the rat mitochondrial cytochrome b gene containing the ochre termination codon

The nucleotide sequences of the genes for cytochrome b and three potential transfer RNAs (tRNAPro, tRNAThr and tRNAGlu) in cloned rat mitochondrial DNA were determined. The derived amino acid sequence of the cytochrome b protein from the light strand indicated that the C-terminal amino acid is asparagine and the ochre termination codon is encoded in the DNA, in contrast to the the lack of termination codon in the reading frame of human [Anderson et al., Nature 290 (1981) 457] or mouse [Bibb et al., Cell 26 (1981) 167] mitochondrial DNA. The first ATG codon of the cytochrome b gene was spaced five nucleotides from the 5'-end of the tRNAGlu gene on the heavy strand. There was a single nucleotide spacing between the termination codon of the cytochrome b gene and the 5' end of the tRNAThr gene in the light strand. There was also a single nucleotide spacing between the 3'-end of the tRNAThr gene and the 3'-end of the tRNAPro gene on the heavy strand. The amino acid and nucleotide sequences of the cytochrome b genes of mammals and yeast [Nobrega and Tzagoloff, J. Biol. Chem. 255 (1980) 9828] were compared to reveal structural differences in two very different species. At the same time, amino acid substitutions in particular regions of the mammalian gene corresponding to the exon-intron boundaries in the yeast gene were noted. These genetic features are discussed in relation to the extreme compression of genetic information in the mammalian mitochondrial genome as related to the evolution of the gene organization and its sequence.     

ABC1, a novel yeast nuclear gene has a dual function in mitochondria: it suppresses a cytochrome b mRNA translation defect and is essential for the electron transfer in the bc 1 complex.

We have cloned and sequenced a novel yeast nuclear gene ABC1 which suppresses, in multicopy, the cytochrome b mRNA translation defect due to the nuclear mutation cbs2-223. Analysis of the ABC1 gene shows that it is weakly expressed, it could code for a protein of 501 amino acids which has a typical presequence of a protein imported into mitochondria and which does not display a strong similarity to any known protein. Inactivation of the ABC1 gene is not lethal to the cell but leads to a respiratory defect: no oxygen uptake and no growth on non-fermentable media. A total absence of NADH-cytochrome c oxidoreductase and succinate-cytochrome c oxidoreductase activities concomitant with the presence of specific dehydrogenases, suggests a block in the bc 1 segment of the respiratory chain. However, all the cytochromes are spectrally detectable. Cytochrome b is quite efficiently reduced while cytochromes c1 and c are not. The function of ABC1 in the suppression of a defect in apocytochrome b mRNA translation and in the activity of the bc1 complex suggests that the ABC1 protein would be a novel chaperonin involved both in biogenesis and bioenergetics of mitochondria.     

Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase

The cyt-2-1 mutant of Neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (Mitchell, M.B., Mitchell, H.K., and Tissieres, A. (1953) Proc. Natl. Acad. Sci. U.S.A. 39, 606-613; Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W., and Neupert, W. (1988) J. Biol. Chem. 263, 9388-9394). By rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a N. crassa genomic library using sib selection. Analysis of the DNA sequence of the cyt-2+ gene revealed an open reading frame of 346 amino acids that has homology to the yeast cytochrome c heme lyase. The open reading frame is interrupted by two short introns. Codon usage and Northern hybridization analysis suggest that the cyt-2 gene is expressed at low levels. The cyt-2-1 mutant allele was cloned from a partial cyt-2-1 gene bank using the wild-type gene as a probe. Sequence analysis of the mutant gene revealed a 2-base (CT) deletion that alters the reading frame for 21 codons before generating an early stop codon in the protein-coding sequence. It was previously suggested that the cyt-2-1 mutation inactivates one of two regulatory circuits controlling the production of cytochrome aa3. The finding that the cyt-2-1 mutation affects the coding sequence for cytochrome c heme lyase provides a direct explanation for the deficiency of cytochrome c in the mutant and suggests that the lack of cytochrome aa3 is a regulatory response to the deficiency of cytochrome c.     

Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme. Cloning, sequencing, and characterization of the nuclear-encoded gene

The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.     

Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex. An intron-containing gene with a conserved sequence occurring in the intron of COX4

A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9. QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471. The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2. The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron. The intron is 213 base pairs long and contains previously reported 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing. The splice junctions, as well as the 5' end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9. In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4. The deduced amino acid sequence of the yeast subunit 9 is 39% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex. If conservative substitutions are allowed for, the two proteins are 56% similar. The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex. Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted. Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b. When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra. These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Yeast omnipotent supressor SUP1 (SUP45): nucleotide sequence of the wildtype and a mutant gene.

The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined. The gene codes for a protein of 49 kD. The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part. The codon usage differs significantly from that of yeast ribosomal protein genes. However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S. cerevisiae has been found. A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria. The hypothetical function of this protein in control of translational fidelity is discussed.     

Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein

We have previously reported the isolation of the gene coding for a 25-kDa polypeptide present in a purified yeast QH2:cytochrome c oxidoreductase preparation, which was thus identified as the gene for the Rieske iron-sulphur protein [Van Loon et al. (1983) Gene 26, 261-272]. Subsequent DNA sequence analysis reported here reveals, however, that the encoded protein is in fact manganese superoxide dismutase, a mitochondrial matrix protein. Comparison with the known amino acid sequence of the mature protein indicates that it is synthesized with an N-terminal extension of 27 amino acids. In common with the N-terminal extensions of other imported mitochondrial proteins, the presequence has several basic residues but lacks negatively charged residues. The function of these positive charges and other possible topogenic sequences are discussed. Sequences 5' of the gene contain two elements that may be homologous to the suggested regulatory sites, UAS 1 and UAS 2 in the yeast CYC1 gene [Guarente et al. (1984) Cell 36, 503-511]. The predicted secondary structures in manganese superoxide dismutase appear to be very similar to those reported for iron superoxide dismutase, suggesting similar three-dimensional structures. Making use of the known three-dimensional structure of the Fe enzyme, the Mn ligands are predicted.