Network of interactions between unlinked genes: synergistic and antagonistic regulation of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 synthesis]

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.     

Formation of composite iso-cytochromes c by recombination between non-allelic genes of yeast

We present evidence that two non-allelic genes, located on two non-homologous chromosomes in the yeast Saccharomyces cerevisiae, recombine and in this process generate new composite genes containing portions of both genes. The two genes CYC1 and CYC7 encode, respectively, iso-1-cytochrome c and iso-2-cytochrome c; CYC1 is located on the right arm of chromosome X and CYC7 is located on the left arm of chromosome V. The coding regions of CYC1 and CYC7 and the corresponding iso-1-cytochrome c and iso-2-cytochrome c are approximately 80% homologous. Composite genes were uncovered among revertants of certain but not all cyc1 mutants lacking iso-1-cytochrome c; composite genes were observed in most revertants from the low-reverting strains cyc1-11, cyc1-136 and cyc1-158, and in low proportions of the revertants from the typically reverting strains cyc1-94 and cyc1-156. Protein analysis of 14 composite iso-cytochromes c and DNA sequencing of five composite genes indicated that recombinational events produced replacements of central portions of the cyc1 gene with a corresponding segment from the wild-type CYC7⁺ gene. The replacements varied in length from 13% to 61% of the translated portion of the CYC1 locus. The formation of composite genes occurred spontaneously at very low frequencies and at low but enhanced frequencies after treatments with mutagens including ultraviolet light, ethylmethane sulfonate, methylmethane sulfonate and nitrous acid. Genetic tests indicated that composite genes are formed mitotically by a conversion-like event in which the wild-type CYC1⁺ allele remains intact. Recombination between non-allelic genes can lead to identical sequences at different loci and to diverse composite genes. These results support the indirect evidence from other eukaryotic systems that non-allelic genes with extensive but not complete homology recombine during evolution.     

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.     

Mutants of Yeast Defective in Iso-1-Cytochrome c

A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cyc1 locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cyc1 mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressors, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cyc1-1, all of the mutants appeared to contain single-site mutations that could be assigned to at least 35 different sites within the gene. The cyc1 mutants either completely lacked iso-1-cytochrome c or contained iso-1- cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyc1 mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cyc1 mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.     

Cytochrome oxidase assembly does not require catalytically active cytochrome C

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.     

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

Deletions of the Iso-1-Cytochrome c and Adjacent Genes of Yeast: Discovery of the OSM1 Gene Controlling Osmotic Sensitivity

Some of the deletions in the yeast Saccharomyces cerevisiae that encompass the CYC1 gene, which determines iso-1-cytochrome c, extend into the OSM1 gene, causing inhibition of growth on hypertonic media, and into the RAD7 gene, causing sensitivity to UV light. Two deletions (cyc1--363 and cyc1--367) encompass only the CYC1 gene, two deletions (cyc1--366 and cyc1--368) encompass the CYC1 and OSM1 genes, three deletions (cyc1--1, cyc1--364 and cyc1--365) encompass the CYC1, OSM1 and RAD7 genes, while none of the deletions extend into the closely linked SUP4 gene.     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

Yeast mutants defective in iso-2-cytochrome c.

The structural gene CYC7 for yeast iso-2-cytochrome c was previously identified by isolating a mutant, cyc7-1-1, totally lacking iso-2-cytochrome c and demonstrating that revertants of this mutant contained iso-2-cytochrome c with an altered primary structure (Downie et al., 1977). In this paper we describe a variety of different types of mutants that completely or partially lack iso-2-cytochrome c due to mutations in either the structural gene, CYC7, or unlinked “regulatory” genes. The iso-2-cytochrome c-deficient mutants were isolated by benzidine staining of over 3 × 10⁵ colonies from ?^? strains (cytoplasmic petites) that lacked iso-1-cytochrome c due to the deletion cyc1-1 and that contain abnormally high levels of iso-2-cytochrome c due to a chromosomal translocation, CYC7-1, adjacent to the normal structural gene CYC7 +. The cytochrome c content of mutants not staining with the benzidine reagents was estimated by low temperature spectroscopy, and 139 mutants containing significantly decreased levels of iso-2-cytochrome c were analyzed genetically by complementation with previously identified cyc mutants. In this way 50 mutants at the cyc2 and cyc3 loci were identified along with a group of 62 mutants of the structural gene cyc7. The different types of mutants of the structural gene which were uncovered and which were more or less anticipated included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA or UAG suppressors, those that lacked iso-2-cytochrome c but had increased levels after growth at lower temperatures, and those that exhibited visibly altered c_a absorption bands of iso-2-cytochrome c. Iso-2-cytochrome c mutants with altered primary structures were obtained from intragenic revertants of several of these mutants, confirming our earlier conclusion that cyc7 is the structural gene. In addition we observed an unexpected class of mutants that lacked iso-2-cytochrome c when in the ?^? state but contained approximately the CYC7-1 parental level when in the ?⁺ state. Two of these mutants, cyc7-1-47 and cyc7-1-49, were shown to contain altered iso-2-cytochromes c. The different contents of the abnormal iso-2cytochromes c suggest that cytochrome c has different environments in ?⁺ and ?^? mitochondria and that the ?⁺ condition may stabilize certain altered proteins.     

A Mutator Affecting the Region of the Iso-1-Cytochrome c Gene in Yeast

The mutator gene DEL1 in the yeast Saccharomyces cerevisiae causes a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYC1, which determines the structure of iso-1-cytochrome c; RAD7, which controls UV sensitivity; and OSM1, which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments. The multisite mutations arise at a rate of approximately 10(-5) to 10(-6) per cell per division in DEL1 strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cyc1 mutants at a low rate of approximately 10(-8) to 10(-9). No evidence for multisite mutations was obtained upon analysis of numerous spontaneous ade1, ade2, met2 and met15 mutants isolated in a DEL1 strain. DEL1 appears to be both cis- and trans-dominant. The location of the DEL1 gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself.     

CYP3 a likely candidate for the structural gene of ISO-2 cytochrome C in Saccaromyces cerevisiae.

Mutations specific for iso-2 cytochrome c were obtained in strains bearing a deletion of the structural gene of iso-1-cytochrome c. In this genetic context the mutations entail an inability to grow on glycerol. One of these mutants was shown to have a modified iso-2-cytochrome c as witnessed by its lack of stability and modified chromatographic behaviour. Genetic studies showed the mutations to be allelic to the mutation cyp3-15 previously identified by Clavilier $\text{et al.}$ (1) as a specific enhancer of iso-2-cytochrome c synthesis. The simplesthypothesis to explain the results is that the CYP3 locus is the structural gene for iso-2-cytochrome c.