Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c.
Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.
Dumont
M E
ME
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Ernst
J F
JF
Sherman
F
F
D001059:Apoproteins / Q000235:genetics*; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics*; D045304:Cytochromes c; D006418:Heme / Q000378:metabolism*; D008190:Lyases / Q000235:genetics*; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D001059:Apoproteins; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase