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.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

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.     

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.     

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.     

Structural gene for yeast iso-2-cytochrome c.

Protein analysis and genetic studies have led to the identification of the structural genes of iso-1-cytochrome c and iso-2-cytochrome c, which constitute, respectively, 95% and 5% of the total amount of cytochrome c in the yeast Saccharomyces cerevisiae. The structural gene CYC1 for iso-1-cytochrome c was previously identified by Sherman et al. (1966) and the structural gene CYC7 for iso-2-cytochrome c is identified in this investigation. A series of the following mutations were selected by appropriate procedures and shown by genetic tests to be allelic: CYC7+ →CYC7-1 →cyc7-1-1 →CYC7-1-1-A, etc., where CYC7 + denotes the wild-type allele determining iso-2-cytochrome c; CYC7-1 denotes a dominant mutant allele causing an approximately 30-fold increase of iso-2-cytochrome c with a normal sequence, and was used as an aid in selecting deficient mutants; cyc7-1-1 denotes a recessive mutant allele causing complete deficiency of iso-2-cytochrome c; and CYC7-1-1-A denotes an intragenic revertant having an altered iso-2-cytochrome c at the same level as iso-2-cytochrome c in the CYC7-1 strains. The suppression of cyc7-1-1 with the known amber suppressor SUP7-a indicated that the defect in cyc7-1-1 was an amber (UAG) nonsense codon. Sequencing revealed a single amino acid replacement of a tyrosine residue for the normal glutamine residue at position 24 in iso-2-cytochrome c from the suppressed cyc7-1-1 strain and also in five revertants of cyc7-1-1, of which three were due to extragenic suppression and two to intragenic reversion. The nature of the mutation that elevated the level of normal iso-2-cytochrome c in the CYC7-1 strain was not identified, although it occurred at or very near the CYC7 locus but outside the translated portion of the gene and it may be associated with a chromosomal aberration. Genetic studies demonstrated that CYC7 is not linked to CYC1, the structural gene for iso-1-cytochrome c.     

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.     

Genetic analysis of yeast iso-1-cytochrome c structural requirements: suppression of Gly6 replacements by an Asn52----Ile replacement.

Gly6 (vertebrate numbering system) is an evolutionarily invariant amino acid located in an electron-dense region of cytochrome c. Serine, cysteine, and aspartic acid replacements of Gly6 abolished yeast iso-1-cytochrome c function, presumably by destabilizing the mature forms of the altered proteins (1). Here we report that genetic reversion analysis of these mutants has uncovered a single base-pair substitution, encoding an Asn52----Ile replacement, that suppresses all three position 6 defects, as well as a Gly6....Gly29----Ser6....Ser29 double replacement. In each case the suppressor restored at least partial function to the altered iso-1-cytochromes c, with the Sera6....Ile52 protein being nearly indistinguishable from the normal protein. The suppressor also affected otherwise normal iso-1-cytochrome c, enhancing the in vivo amount of the protein by about 20%. While this work was in progress, Das et al. (1989, Proc. Natl. Acad. Sci. USA 86, 496-499) uncovered Ile52 as a suppressor of single Gly29 and His33 replacements in iso-1-cytochrome c. The ability of Ile52 to suppress amino acid replacements at three different sites, and its effect in isolation from the primary mutations, defines Ile52 as a global suppressor of specific iso-1-cytochrome c structural defects. These data suggest that position 52 plays a critical role in the folding and/or stability of iso-1-cytochrome c.     

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.     

An extensive deletion causing overproduction of yeast iso-2-cytochrome c

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event.     

Structure determination and analysis of yeast iso-2-cytochrome c and a composite mutant protein.

As part of a study of protein folding and stability, the three-dimensional structures of yeast iso-2-cytochrome c and a composite protein (B-2036) composed of primary sequences of both iso-1 and iso-2-cytochromes c have been solved to 1.9 A and 1.95 A resolutions, respectively, using X-ray diffraction techniques. The sequences of iso-1 and iso-2-cytochrome c share approximately 84% identity and the B-2036 composite protein has residues 15 to 63 from iso-2-cytochrome c with the rest being derived form the iso-1 protein. Comparison of these structures reveals that amino acid substitutions result in alterations in the details of intramolecular interactions. Specifically, the substitution Leu98Met results in the filling of an internal cavity present in iso-1-cytochrome c. Further substitutions of Val20Ile and Cys102Ala alter the packing of secondary structure elements in the iso-2 protein. Blending the isozymic amino acid sequences in this latter area results in the expansion of the volume of an internal cavity in the B-2036 structure to relieve a steric clash between Ile20 and Cys102. Modification of hydrogen bonding and protein packing without disrupting the protein fold is illustrated by the His26Asn and Asn63Ser substitutions between iso-1 and iso-2-cytochromes c. Alternatively, a change in main-chain fold is observed at Gly37 apparently due to a remote amino acid substitution. Further structural changes occur at Phe82 and the amino terminus where a four residue extension is present in yeast iso-2-cytochrome c. An additional comparison with all other eukaryotic cytochrome c structures determined to date is presented, along with an analysis of conserved water molecules. Also determined are the midpoint reduction potentials of iso-2 and B-2036 cytochromes c using direct electrochemistry. The values obtained are 286 and 288 mV, respectively, indicating that the amino acid substitutions present have had only a small impact on the heme reduction potential in comparison to iso-1-cytochrome c, which has a reduction potential of 290 mV.     

Rational design of a more stable yeast iso-1-cytochrome c.

Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c. We have used a coordinated approach, combining the known functional and structural properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with the goal of selectively increasing the stability of the protein. The two redox forms of the native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed by differential scanning calorimetry (DSC). The relative stability, expressed as the difference in the Gibb's free energy of denaturation at a given temperature between the native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins. In both oxidation states, the mutant proteins C102T, T69E/C102T, T96A/C102T, and T69E/T96A/C102T were more stable than the wild-type protein, respectively. The increased stability of the mutant proteins is proposed to be due to the removal of a rare surface cysteine and the stabilization of two distorted alpha-helices.     

Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein

Site-directed mutagenesis has been used to change the codon for cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c to a threonine codon. The resulting protein is active in vivo, is methylated as in the wild-type protein and has optical properties indistinguishable from those of the wild-type protein. The threonine-107 iso-1-cytochrome c demonstrated fully reversible electrochemical behaviour and a mid-point reduction potential of 272 mV versus NHE. In addition, this mutant does not demonstrate a tendency to autoreduce or to dimerize as does the wild-type protein. These properties of the threonine-107 mutant establish that it will provide a useful background in which to make subsequent mutations for mechanistic and physical studies of yeast iso-1-cytochrome c.     

A Chromosomal Translocation Causing Overproduction of Iso-2-Cytochrome c in Yeast

The CYC7–1 mutation in the yeast Saccharomyces cerevisiae causes the production of approximately 30 times the normal amount of iso-2-cytochrome c. Genetic analysis established that the CYC7–1 mutation is a reciprocal translocation involving the left arm of chromosome V and the right arm of chromosome XVI. The chromosome V arm was broken adjacent to the gene CYC7, which determines the primary structure of iso-2-cytochrome c, and this fragment containing the CYC7 gene was joined to the segment of chromosome XVI. It appears as though the elevation of iso-2-cytochrome c is caused by an abnormal controlling region adjacent to the structural region of the CYC7 gene.     

Mutants of yeast initiating translation of Iso-1-cytochrome c within a region spanning 37 nucleotides

We used a specially constructed strain, cyc1–345, of the yeast Saccharomyces cerevisiae to isolate revertants that initiated translation of iso-1-cytochrome c at various sites along an extended region of the mRNA. Normal amounts of iso-1-cytochrome c occurred when translation initiated at the abnormal sites corresponding to amino acid positions ?3, ?2, 3 and 5, as well as the normal position ?1; 20% of the normal amounts occurred when translation initiated at the abnormal position 9. These results with cyc1–345 revertants indicate that translation of iso-1-cytochrome c can initiate with the normal efficiency at any site within the region spanning 25 nucleotides. Furthermore, because the lower amount of the short iso-1-cytochrome c in the mutant initiating at position 9 may not necessarily reflect an inefficiency of translation, we believe that translation can initiate with normal or near-normal efficiencies at any site within a 37 nucleotide region, and presumably at any site preceding and following that of the normal initiation codon. These results establish that there is no absolute requirement for a particular sequence 5′ to the initiation codon, and are consistent with our previous suggestion that translation starts at the AUG codon closest to the 5′ end of the mRNA.     

Applications of synthetic oligodeoxyribonucleotides to problems in molecular biology

The applications of synthetic oligodeoxyribonucleotides to problems in molecular biology described in this article are those where the oligodeoxyribonucleotide is a probe for a specific region of a nucleic acid. This includes the isolation of the iso-1-cytochrome c gene of yeast; the sequence determination of RNAs and DNAs including regions of double-stranded DNA; the introduction of defined site-specific point mutations into bacteriophage OX174 and in the in vitro selection of mutant DNA from a mixture with wild-type DNA.