Studies on the lipid dependency and mechanism of the translocation of the mitochondrial precursor protein apocytochrome c across model membranes.
The lipid dependency of apocytochrome c binding to model membranes and of the translocation of the precursor protein across these membranes was studied by using large unilamellar, trypsin-containing vesicles. These vesicles were improved with respect to those used in a previous article (Rietveld, A., and de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6706), in the sense that a lower amount of trypsin was enclosed. In mixed egg phosphatidylcholine/bovine brain phosphatidylserine vesicles, both the Kd of apocytochrome c binding (about 20 microM) and the number of phosphatidylserine molecules interacting with the protein was found to be constant. When the phosphatidylserine fraction in the vesicles is more than 15-30% apocytochrome c addition results in the exposure of (a part of) the protein to the internal, trypsin-containing vesicle medium, which process we conceive as a translocation event. Also the interaction of apocytochrome c with vesicles composed of phosphatidylcholine and another acidic phospholipid in a 1:1 ratio, leads to the translocation of the protein across the model membrane. The affinity of this binding was found to be in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. By varying the lipid composition of the vesicles, it could be demonstrated that the translocation requires a fluid bilayer. In addition, protein specificity was shown for the translocation process. Although apocytochrome c-lipid interaction causes vesicle aggregation, fusion by lipid mixing could not be detected. Due to the apocytochrome c-lipid interaction also, protein aggregates and oligomers have been formed. These results will be discussed in the light of a model for translocation of a precursor protein across a model membrane. The relevance for the mitochondrial system will also be discussed.
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D001692:Biological Transport; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D045304:Cytochromes c; D006736:Horses; D008051:Lipid Bilayers / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D001059:Apoproteins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011498:Protein Precursors; D045304:Cytochromes c; D014357:Trypsin