A method of directed random mutagenesis of the yeast chromosome shows that the iso-1-cytochrome c heme ligand His18 is essential.

A method to perform site-directed random mutagenesis directly in the yeast chromosomal DNA at the iso-1-cytochrome c-encoding gene locus (CYC1) is described. To test the effectiveness of the random mutagenesis procedure, the heme ligand His18 was mutated to Ala (H18A), rendering cytochrome c (Cyc) nonfunctional. Random mutagenesis was performed by transforming yeast cells with a synthetic oligodeoxyribonucleotide (oligo) that randomizes the codon for His18. The transformed cells were then selected for reversion to a functional Cyc on selective media. Ten functional mutants were recovered, all of which had integrated the synthetic oligo. Sequencing showed that five of the recovered mutants carried the His codon, CAU, and five mutants contained the His codon, CAC. Because Arg had previously been found as a heme ligand, this mutant was produced by standard techniques and integrated into the yeast chromosome. These yeast did not produce a holo cytochrome c that was detectable by low-temperature spectroscopy. To develop a selection for nonfunctional Cyc, competent yeast (which lack the ability to synthesize tryptophan) were cotransformed with a plasmid carrying the TRP1 gene and the random oligo, and were plated on media lacking tryptophan. Of the 1200 colonies that grew, 120 tested negative for the integration of the random oligo, demonstrating that this particular selection for nonfunctional protein is not feasible. A method is thus described for directed, random mutagenesis directly in the yeast chromosome that can be used to probe structure/function relationships in Cyc. Only His can act as a heme ligand at position 18, using the functional selection described here.

Loop replacement and random mutagenesis of omega-loop D, residues 70-84, in iso-1-cytochrome c.

To study the role of omega loop D, residues 70-84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.

Structure, function, and temperature sensitivity of directed, random mutants at proline 76 and glycine 77 in omega-loop D of yeast iso-1-cytochrome c.

Residues 75-78 form a tight turn within Omega-loop D in Saccharomyces cerevisiae iso-1-cytochrome c. Directed, random mutagenesis of invariant residues proline 76 and glycine 77 in this turn were analyzed for the in vivo functionality and level of protein within the cell. All proteins, except Pro76Val, also exhibit a significant decrease in intracellular cytochrome c levels, ranging from 15% to 80% of wild type. Furthermore, all isolated mutant strains, except the one expressing Pro76Val, exhibit a significant decrease in growth on lactate medium, suggesting that the variant cytochromes are much less functional than wild type. This requirement for protein function is clearly the cause for the strict invariance of these residues in eukaryotic cytochromes c. Seven proteins with mutations just at Pro76 were purified and studied by circular dichroism spectroscopy. All proteins with mutations at Pro76 exhibit melting temperatures about 7 degreesC less than that of the wild-type protein, suggesting that mutation of Pro76 affects the entropy of the denatured state. It is proposed that the functional significance of Pro76 and Gly77 is the requirement for a type II (betagammaL) beta-turn in this loop, the conformation of which requires a glycine at the third position, and that a change occurs in this turn conformation upon a change in the redox state of the protein.