Sequence requirements for mitochondrial import of yeast cytochrome c.
Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.
Wang
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Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Dumont
M E
ME
Sherman
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F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D004171:Diploidy; D006238:Haploidy; D006418:Heme / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003574:Cytochrome c Group; D007527:Isoenzymes; D009841:Oligonucleotides; D006418:Heme