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.     

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.     

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.     

Primary site and second site revertants of missense mutants of the evolutionarily invariant tryptophan 64 in iso-1-cytochrome c from yeast.

The three missense mutants cyc1-132, cyc1-166 and cyc1-189 in the yeast Saccharomyces cerevisiae contain nonfunctional and thermolabile iso-1-cytochromes c and have different replacements of the tryptophan at position 64 which corresponds to the invariant tryptophan residue found in cytochromes c from all eukaryotic species. The cyc1-166 and cyc1-189 mutants contain single replacements of, respectively, serine 64 and cysteine 64, while the cyc1-132 mutant contains a double replacement of glycine 64 and alanine 65 instead of the normal tryptophan 64 and aspartic acid 65. Twenty-three intragenic revertants having at least partially functional iso-1-cytochromes c arose from these three missense mutants by single amino acid replacements of either tryptophan, phenylalanine, tyrosine or leucine at position 64, or by second-site replacements in which the mutant residues at position 64 are retained and the normal serine 45 is replaced by phenylalanine 45. Specific activities of the iso-1-cytochromes c were estimated by growth of strains on lactate medium and are as follows, in terms of the normal, for iso-1-cytochromes c altered specifically in the ways shown: 100% for phenylalanine 64; 25% for tyrosine 64; between 0 and 25% for leucine 64; 100% for phenylalanine 45, cysteine 64; 25% for phenylalanine 45, serine 64; between 0 and 25% for phenylalanine 45, glycine 64, alanine 65; and 0% for serine 64, for cysteine 64, and for glycine 64, alanine 65 iso-1-cytochromes c. The results demonstrate that small residues of glycine, serine, and cysteine at position 64 are incompatible with function; they imply that many of the 10 amino acids accessible by single base-pair substitution but not observed in primary site revertants also are incompatible with function; and they show that large hydrophobic residues of phenylalanine, leucine, and tyrosine at position 64 are capable of restoring at least partial function. The second site revertants indicate that deleterious effects of the three missense mutants can be compensated by the introduction of phenylalanine 45, which may occupy space normally filled by tryptophan 64. Altered shapes of Calpha-band spectra and at least partial instability were characteristics of all iso-1-cytochromes c found lacking tryptophan 64. Apparently, the principal role of the invariant tryptophan is stabilization of the active protein structure, by providing a large hydrophobic group at the proper location.     

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.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

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.     

Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.     

Specific induction of transitions and transversions of G-C base pairs by 4-nitroquinoline-1-oxide in iso-1-cytochrome c mutants of yeast

The base-pair changes induced by the highly carcinogenic agent, 4-nitroquinoline-1-oxide, have been determined from the reversion rates of defined tester strains and from the amino acid replacements of revertant iso-1-cytochromes c. The mutant codons and the base-pair changes of reverse mutations of 14 cyc1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc1 mutants, which were used as tester strains, included nine mutants with altered AUG initiation codons, an ochre (UAA) mutant, an amber (UAG) mutant and three frameshift mutants (Stewart et al., 1971,1972; Stewart &; Sherman, 1972,1974; Sherman &; Stewart, 1973). NQO² induced a high rate of reversion in the initiation mutant cyc1-131, the only mutant in the group which reverts to normal iso-1-cytochrome c by a G · C → A · T transition. In addition, NQO produces a significant rate of reversion of all cyc1 mutants which revert by G · C transversions, e.g. the amber (UAG) mutant and the initiation mutants containing AGG, and probably CUG mutant codons. It did not revert the ochre mutant which contains no G · C base pairs. Ten NQO-induced revertants of the amber mutant cyc1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc1-131 and cyc1-133 initiation mutants all contained the expected normal iso-1-cytochrome c. The structures of these iso-1-cytochromes c and the pattern of reversion of the tester strains indicate that base-pair substitutions arise at G · C base pairs which are the site of NQO attack. Thus NQO induces G · C → A · T transitions, G · C → T · A transversions and possibly G · C → C · G transversions. Because of its mode of action, NQO may be useful in less-defined systems for identifying G · C base pairs in mutant codons.     

Amino acid replacements of the evolutionarily invariant tryptophan at position 64 in mutant forms of iso-1-cytochrome c from Saccharomyces cerevisiae

Tryptophan located at position 59 in vertebrate cytochromes c and at position 64 in yeast iso-1-cytochrome c is an evolutionarily invariant residue that is believed to be essential to the operation of the cytochrome c molecule. We show that this residue is replaced in at least partially functional iso-1-cytochromes c from cyc1 revertants of the yeast Saccharomyces cerevisiae. Tryptophan, tyrosine and leucine are found at position 64 in the revertants from the cyc1-84 mutant, confirming the genetic evidence (Sherman et al., 1974) that the mutant contains an UAG nonsense codon and establishing that the site of the mutation corresponds to the normal tryptophan 64. In a revertant from the cyc1.189 mutant, position 64 is occupied by a residue of phenylalanine. All three altered proteins are unstable, implying that tryptophan 64 has an essential and unique role for maintaining the normal structure of the cytochrome c molecule. In addition the iso-1-cytochrome c with leucine 64 and tyrosine 64 have greatly reduced biological activities, while iso-1-cytochrome c with the phenylalanine replacement has at least 20% of the wild-type activity or more. It remains uncertain whether the reduced specific activities are due to distorted tertiary structures or due to the specific lack of the tryptophan residue that may also have a direct functional role.     

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.     

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.     

Confirmation of UAG as a nonsense codon in bakers' yeast by amino acid replacements of glutamic acid 71 in iso-1-cytochrome c

The yeast mutant cy1–76 is more than 99% deficient in iso-1-cytochrome c. Twelve intragenic revertants of cy1–76 have approximately normal amounts of iso-1-cytochromes c, which are altered by replacement of glutamic acid 71 with either tryptophan, leucine, tyrosine, serine, glutamine or lysine. It is concluded that position 71 in functioning iso-1-cytochrome c can be radically varied, and that the defect in cy1–76 is a nonsense codon, UAG, corresponding to position 71.Tryptophan is the replacement in 4 of the 12 revertants of cy1–76. Tryptophan is similarly abundant as a replacement of lysine 9 in the previously studied 42 revertants ofcy1–179, but is not a replacement in the 45 previously studied revertants of cyl-9. Since amino acid replacements indicate that either UAA or UAG nonsense mutations occur in all three mutants, these new results confirm the previously recognized distinction between the two nonsense codons: one, evidently UAG, can be reverted to a tryptophan codon, while the other, apparently UAA, cannot; apparently UGA does not encode tryptophan in yeast.     

Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.