Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae.

Abstract

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.

Cytochrome c Heme Lyase Activity of Yeast Mitochondria

“…The concentration of cytochrome c was determined by its absorption at 550 nm after reduction with sodium dithionite, using an extinction coefficient of 27.6 mM Ϫ1 cm Ϫ1 (30). Isogenic Yeast Strains-Yeast strains B-7553 (MATa CYC1 ϩ cyc7-⌬::cyh2 ura3-52 his3-⌬1 leu2-3, 112 trp1-289 cyh2) (31), B-8025, which is the same as B-7553 with CYC3 deleted, B-8132, which is the same as B-7553 with CYC2 deleted (31), and A42B, which is B-7553 that has been transformed with plasmid pAA268 containing CYC3 under control of the actin promoter on a multicopy plasmid with URA3 as a marker (11,18), were all kindly provided by Dr. Mark E. Dumont. CYC1 and CYC7 are the structural genes for yeast iso-1 and iso-2 cytochrome c, respectively, so the strain B-7553 is with normal iso-1 cytochrome c but without iso-2 cytochrome c. CYC3 encodes the yeast cytochrome c heme-lyase (18), so the strain A42B is with overexpressed heme lyase, whereas B-8025 is without heme lyase.…”

“…Although the genes for cytochrome c heme lyase of Saccharomyces cerevisiae (18) and Neurospora crassa (19) have been cloned and the role of the enzyme in the import of apocytochrome c has been described, as given above (11, 20 -24), the two systems in use for the in vitro assay of heme lyase activity are both complicated and inconvenient and either of low efficiency (20,22) or require a large amount of mitochondria and apoprotein (18,24,(25)(26)(27)(28). To study heme lyase and its relation to the high affinity binding of the apoprotein to mitochondria, a simple and efficient HPLC 2 method was developed to assay the activity of the enzyme, using biosynthetically made, pure apocytochrome c and yeast mitochondria.…”

AtCCMH, an essential component of thec-type cytochrome maturation pathway inArabidopsismitochondria, interacts with apocytochromec

“…Earlier studies in yeast (system III) showed cytochrome c and cytochrome c 1 heme lyases (CCHL and CCHL 1 ) to be the central players in the formation of mitochondrial cytochrome c and c 1 (4,5). Orthologs of CCHL genes are found in nuclear genomes of vertebrates, invertebrates, and some green algae (i.e., Chlamydomonas reinhardtii), suggesting that their mitochondrial c-type cytochromes are assembled through system III.…”

Contact sites between inner and outer membranes

“…The precursor protein, apocytochrome c, inserts into the outer membrane without the aid of surface receptors or the general insertion protein [28,29]. The enzyme cytochrome c heme lyase that adds heme to the apoprotein on the intermembrane space side represents the only identified component of the import machinery for apocytochrome c [30,31]. Most interestingly, cytochrome c heme lyase appears to be located in or close to contact site regions although all available evidence suggests that there is no direct relation between the import pathway of apocytochrome c and that of precursors using surface receptors.…”