The binding of cytochrome b5 to phospholipid vesicles and biological membranes. Effect of orientation on intermembrane transfer and digestion by carboxypeptidase Y.

A method is described which allows the direct measurement of intermembrane protein transfer. We have used this method to examine the transfer of cytochrome bg from artificial phospholipid vesicles and biological membranes. !l!his method involves the incubation of small, sonicated phospholipid vesicles with either biological membranes or large unilamellar phospholipid vesicles (Enoch, H. G., and Strittmatter, P. (1979) Proc. Natl. Acad Sci. U. S. A. 76, 145-149) and subsequent separation by gel filtration. We have observed cytochrome ba transfer between large and small single bilayer vesicles when cytochrome b5 was bound to preformed egg phosphatidylcholine vesicles. The cytochrome bs of microsomes, however, did not transfer to small vesicles; neither did cytochrome bg reductase nor exogenous, bound cytochrome bs. In fact, no detectable protein was transferred when high salt-washed microsomes were mixed with small, sonicated vesicles. Similar results were obtained using mitochondria and nuclear membrane fragments. We conclude that integral membrane proteins in general do not readily undergo intermembrane transfer between biological membranes. The ability of cytochrome b5 to transfer from artificial membranes and not from biological membranes may reflect a difference in the nature of the protein binding. A nontransferable form of cytochrome bg, which may represent the microsomal type of binding, was obtained when cytochrome bg was bound to preformed vesicles of dimyristyl phosphatidylcholine or when cytochrome b5 was bound during the formation of phosphatidylcholine vesicles. A soluble, heme peptide fragment of cytochrome bs was released when vesicles containing cytochrome be in the transferable form were incubated with carboxypeptidase Y. In contrast, the nontransferable form of cytochrome b5 in microsomes and artificial vesicles was not released by carboxypeptidase Y treatment. We conclude that there are at least two possible orientations of cytochrome bs in phospholipid bilayers, and that these orientations result in either hindered or rapid intermembrane transfer.