Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein. We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c. Mitochondrial extracts from cyc3‐ mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity. The CYC3 gene was cloned by functional complementation of a cyc3‐ mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3. An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length. CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.

The cyc1‐512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3′ untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1–512 mRNA contained many discrete 3′ termini at certain sites, ranging from the wild‐type position to over 2000 nucleotides (nt) downstream. The cyc1–512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base‐pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG…TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3′ endpoints, apparently identical to those seen in the cyc1–512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1–512 mRNAs. The capacity of these three signals to form 3′ ends was confirmed with sequences constructed by site‐directed mutagenesis. We therefore suggest that the production of 3′ termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3′ mRNA termini, as represented by the cyc1–512 termini. The second type of element, which includes TAG…TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3′ preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)

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We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso‐l‐cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso‐l‐cytochrome c was facilitated by the recently resolved three dimensional structure of iso‐l‐cytochrome c [(1987) J. Mol. Biol. 199, 295–314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso‐l‐cytochrome c function appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso‐l‐cytochrome c.

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