Reversible import of apocytochrome c into mitochondria.
35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.
Hakvoort
T B
TB
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Sprinkle
J R
JR
Margoliash
E
E
D000409:Alanine; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D003545:Cysteine; D003574:Cytochrome c Group / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045304:Cytochromes c; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D008930:Mitochondria, Liver / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012694:Serine; D014158:Transcription, Genetic
D001059:Apoproteins; D003574:Cytochrome c Group; D012694:Serine; D045304:Cytochromes c; D003545:Cysteine; D000409:Alanine