Identification of Missense Mutants by Amino Acid Replacements in Iso-l-cytochrome c from Yeast

Altered peptides from chymotryptic digests of iso-1-cytochromes c that were obtained from two revertants of the cyc1–6 mutant, one revertant of cyc1–10 mutant, and one revertant of the cyc1–15 mutant have been isolated and their amino acid sequences established. The single amino acid replacements, serine and threonine at position 12 in two of the revertant iso-1-cytochromes c, indicated that cyc1–6 is a missense mutant which contains a residue of proline at position 12 instead of the normal residue of alanine. The RNA codons for proline differ from the RNA codons of alanine, serine, and threonine by one base, indicating that the cyc1–6 mutant arose by a G·C → C·G transversion and the serine and threonine revertants were formed, respectively, by a G·C → A·T transition and a G·C → T·A transversion. The revertant from cyc1–10 contained a residue of asparagine at position 13 in place of the normal residue of threonine, and the revertant from cyc1–15 contained a residue of isoleucine at position 17 in place of the normal residue of threonine. While it has not been possible to deduce the exact nature of the mutational changes in the cyc1–10 and cyc1–15 mutants since only one altered iso-1-cytochrome c was characterized from each of these cyc1 mutants, it is clear that these replacements are not compatible with mutations by way of any of the known nonsense codons. The complete deficiency of iso-1-cytochrome c in the cyc1–10 mutant and the amino acid replacement in the revertant could be explained either by frameshift mutations, if the forward and reverse mutations occurred within a single codon, or more likely by a missense mutation that gave rise to a lysine residue at this site which is incompatible for as yet undefined steps in the attachment of the heme group. We have no simple explanation for the cyc1–15 mutation since the site of the amino acid replacement in the revertant is not compatible with the site of the mutation that was determined by genetic fine structure mapping.