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.     

Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast

The eight class I, set 1 super-suppressor genes, SUP2, SUP3, SUP4, SUP5, SUP6, SUP7, SUP8 and SUP11 are not closely linked and map at distinct loci throughout the genome of yeast. Each of these suppressors causes the production of 5 to 10% of the normal amount of iso-1-cytochrome c when it is individually coupled to the ochre (UAA) mutant cy1-2. All eight iso-1-cytochromes c contain a residue of tyrosine at position 20 which corresponds to the site of the ochre codon. Several of these super-suppressors also were shown to act on cy1-9, but at a much lower efficiency. It was shown that iso-1-cytochrome c from one of the suppressed cy1-9 strains contains a tyrosine at position 2, which corresponds to the site of the ochre codon in this mutant. It is suggested that the gene product of the eight super-suppressors is tyrosine transfer RNA.     

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.     

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.     

Deletions and replacements of omega loops in yeast iso-1-cytochrome c

omega (omega)-loops are protein secondary structural elements having small distances between segment termini. It should be possible to delete or replace certain of these omega-loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae were investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different omega-loops were individually deleted, or in which one omega-loop was replaced with five different segments. Deletions encompassing amino acid positions 27-33 and 79-83 either prevented synthesis of the holoprotein, or produced highly labile iso-1-cytochromes c, whereas deletions encompassing positions 42-45 and 48-55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue positions 24-33) with same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso-1-cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.     

Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.     

A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.     

Folding/unfolding kinetics of mutant forms of iso-1-cytochrome c with replacement of proline-71

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. To assign the effects of perturbations at position 71 to steps in the process of protein folding, the kinetic properties of the folding/unfolding reactions of normal protein and the three mutant forms are compared. At pH 6.0, 20 degrees C, fluorescence-detected folding/unfolding kinetics are monitored below, within, and above the equilibrium transition zone by using stopped-flow mixing to perform guanidine hydrochloride concentration jumps. Three kinetic phases are detected for each of the four proteins. The fastest of these phases (tau 3) differs in rate for the wild type and mutant proteins. The remaining kinetic phases (tau 1 and tau 2) have similar rates for all four proteins over the entire range of folding/unfolding conditions. The guanidine hydrochloride dependence of the relative amplitudes of the kinetic phases is complex and is sensitive to the nature of the substituent at position 71: each of the four proteins shows differences in the fraction of folding/unfolding associated with the two fastest rate processes. The results suggest that it is the location of the mutation in the primary structure rather than the nature of the substituent that determines which kinetic step (or steps) is changed in rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanidine hydrochloride induced equilibrium unfolding of mutant forms of iso-1-cytochrome c with replacement of proline-71

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. Treatment of these proteins with a specific sulfhydryl blocking reagent (methyl methanethiosulfonate) to block Cys-102 has allowed investigation of the properties of monomeric forms of the proteins, denoted iso-1-MS. Comparison of the UV-visible absorbance properties (pH 6, 20 degrees C) shows minor differences between the normal Pro-71 iso-1-MS and two of the three mutant proteins. The Val-71 iso-1-MS protein has absorbance properties indistinguishable from those of the normal Pro-71 iso-1-MS protein, but the Ile-71 iso-1-MS and Thr-71 iso-1-MS proteins show reduced intensity of the 695-nm absorbance band and a small shift in the Soret maximum, from 408 nm for the Pro-71 iso-1-MS and Val-71 iso-1-MS proteins to 406 nm for the Thr-71 iso-1-MS and Ile-71 iso-1-MS proteins. Second derivative spectroscopy is used to assess differences in the polarity of the environment of tyrosine residues. The average degree of exposure of tyrosines to solvent is similar in all four proteins: 0.39 for the normal Pro-71 iso-1-MS and Val-71 iso-1-MS proteins; 0.40 for the Ile-71 iso-1-MS protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-sensitive variants of Saccharomyces cerevisiae iso-1-cytochrome c produced by random mutagenesis of codons 43 to 54.

In vitro random mutagenesis within the CYC1 gene from the yeast Saccharomyces cerevisiae was used to produce a library of mutants encompassing codons 43 to 54 of iso-1-cytochrome c. This region consists of an evolutionarily conserved structure within an evolutionarily diverse sequence. The library, on a low-copy-number yeast shuttle phagemid, was introduced into a yeast strain lacking cytochrome c. The ability of transformants harboring a functional cytochrome c to grow on the non-fermentable carbon source glycerol at 30 degrees C and 37 degrees C was used to determine the phenotype of nearly 1000 transformants. Approximately 90% of the missense mutants present in the library give rise to the wild-type phenotype, 7% result in the temperature-sensitive (Cycts) phenotype, and 3% give rise to the non-functional (Cyc-) phenotype. Phagemids from 20 Cycts and 30 Cyc- transformants were subjected to DNA sequence analysis. All the mutations occur within the targeted region. One-third of the mutants from Cyc- transformants and all the mutants from Cycts transformants are missense mutants. The remaining mutants from Cyc- transformants are nonsense or frame-shift mutants. Missense mutations within the codons for Gly45, Tyr46, Thr49, Asn52 or Ile53 alone are sufficient to produce temperature-sensitive behavior both in vivo and in the variant proteins. The deduced amino acid substitutions correlate remarkably well with side-chain dynamics, secondary structure and tertiary structure of the wild-type protein.     

Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase

Amino-terminal processing in the yeast Saccharomyces cerevisiae has been investigated by examining numerous mutationally altered forms of iso-1-cytochrome c. Amino-terminal residues of methionine were retained in sequences having penultimate residues of arginine, asparagine, glutamine, isoleucine, leucine, lysine, and methionine; in contrast, the amino-terminal methionine residues were exercised from residues of alanine, glycine, and threonine and were partially excised from residues of valine. The results suggest the occurrence of a yeast aminopeptidase that removes amino-terminal residues of methionine when they precede certain amino acids. A systematic search of the literature for amino-terminal sequences formed at initiation sites suggests the hypothetical yeast aminopeptidase usually has the same specificity as the amino peptidase from bacteria and higher eukaryotes. Our results and the results from the literature search suggest that the aminopeptidase cleaves amino-terminal methionine when it precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine glutamic acid, isoleucine, leucine, lysine, or methionine. In contrast to the normal iso-1-cytochrome c and in contrast to the majority of the mutationally altered proteins, certain forms were acetylated including the following sequences: acetyl(Ac)-Met-Ile-Arg-, Ac-Met-Ile-Lys, Ac-Met-Met-Asn-, and Ac-Met-Asn-Asn-. We suggest yeast contains acetyltransferases that acetylates these mutant forms of iso-1-cytochromes c because their amino-terminal regions resemble the amino-terminal regions of natural occurring proteins which are normally acetylated. The lack of acetylation of closely related sequences suggest that the hypothetical acetyltransferases are specific for certain amino-terminal sequences and that the 3 amino-terminal residues may play a critical role in determining these specificities.     

Sequence of the yeast iso-1-cytochrome c mRNA

The nucleotide sequence of the yeast iso-1-cytochrome c (CYC1) mRNA is presented. The mRNA was enriched by hybridization to cloned CYC1 DNA attached to a solid matrix: either nitrocellulose filters or diazobenzyloxymethyl cellulose powder. The sequence of the 5'-end of the mRNA was determined by the extension of a CYC1-specific dodecanucleotide primer; the sequence of the 3'-end was determined using a decanucleotide d(pT8-G-A) primer. The CYC1 mRNA begins 61 nucleotides 5' to the AUG initiation codon, extends through the coding sequence to 172 to 175 nucleotides 3' to the UAA termination codon, followed by the poly(A) tail. There are no intervening sequences. Some of the sequences that the CYC1 mRNA shares in common with other eukaryotic mRNAs are discussed.     

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector.

Phe82 is the most studied invariant residue of cytochrome c. However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e. using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector. Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level. Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon. The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast. All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c. However the F82C, F82P, and F82R variants grow at a significantly lower rate. After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified. It is concluded that all 20 amino acids are capable of supporting function. Reasons for the evolutionary invariance of Phe82 are discussed.     

Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c.

Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.     

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.