The importance of the amino terminus of the mitochondrial precursor protein apocytochrome c for translocation across model membranes.
The importance of the various regions of the apocytochrome c molecule and the effect of covalent coupling of the heme group to the protein for the translocation across a model-membrane was studied by using chemically and enzymatically prepared fragments of horse heart apo- and holocytochrome c and model-membranes composed of phosphatidylserine and phosphatidylcholine. Binding experiments showed that fragments of apocytochrome c with the highest net positive charge have the highest affinity for negatively charged large unilamellar phosphatidylserine vesicles. Monolayer experiments demonstrated that the amino-terminal fragments were only able to penetrate into a phosphatidylserine monolayer whereas the carboxyl-terminal fragments, in addition, penetrated into a phosphatidylcholine monolayer although with a lower efficiency. The covalent coupling of the heme group to both a small amino-terminal fragment residue numbers 1-38 and to the entire precursor protein resulted in a marked decrease in the ability to penetrate into a phosphatidylserine monolayer. Translocation experiments with trypsin enclosed in vesicles, showed that only the amino-terminal fragments of the precursor protein and not carboxyl-terminal peptides or the heme-containing fragments of the mature protein were able to cross the bilayer and become digestible by trypsin at the opposite side of the bilayer. Circular dichroism measurements with the various peptides both in an aqueous and lipidic environment were performed to investigate the conformation of apocytochrome c after interaction with model-membranes. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.
Jordi
W
W
Department of Biochemistry, State University of Utrecht, The Netherlands.
Zhou
L X
LX
Pilon
M
M
Demel
R A
RA
de Kruijff
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D007425:Intracellular Membranes / Q000378:metabolism; D008081:Liposomes; D008930:Mitochondria, Liver / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D013367:Submitochondrial Particles / Q000378:metabolism
D001059:Apoproteins; D003574:Cytochrome c Group; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D045304:Cytochromes c