Partial translocation of apocytochrome c across phospholipid bilayer caused by tryptic digestion

Abstract

In contrast to easlier reports of complete translocation, apocytochrome c is found to be only partially translocated across the bilayer of PS vesicles entrapped with trypsin. A 2.5 kDa polypeptide segment of C-terminal area of apocytochrome c (80-104) remains attasched to the bilayer after the rest of the apocytochrome c is digested by internal trypsin. Digestion of vesicle-bound apocytochrome c by externally added trypsin also yielded a protected segment in the C-terminal area (74-104). These observations suggest that apocytochrome c spans the bilayer when it binds to PS vesicles and there is a partial translocation when it is being digested by either external or internal trypsin. Externally added apocytochrome c was hydrophobically labeled with [I] TID and treated with endoproteinase Arg C. The results indicates that the N-terminal area (1-38) penetrates the bilayer. The vesicle-bound apocytochrome c was also digested with aminopeptidase showing that the N-terminal end is exposed to the outside. A dynamic interaction between apocytochrome c and vesicles and a possible partial translocation model are discussed.