Distribution of cytochrome b5 between sonicated phospholipid vesicles of different size.

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose," or transferable, configuration characterized by the ability of the protein to rapidly equilibrate between vesicles. A heterogeneous dispersion of sonicated phosphatidylcholine vesicles, 212 to about 350 A in diameter, was prepared by differential centrifugation. When cytochrome b5 was incubated with these vesicles (1 mol of protein/833 mol of phospholipid, in 0.01 M NaHCO3, 0.1 M NaCl, 10(-4) M EDTA, pH 7.4) and the mixture was subjected to molecular sieve chromatography on Sepharose 2B-CL, the cytochrome b5 was found to elute preferentially with the smaller vesicles. Subsequently, a fresh preparation of heterogeneous vesicles was subfractionated by gel filtration, and the individual fractions were incubated with the protein. Molecular sieve chromatography of these complexes showed that cytochrome b5 favors the smallest over the largest vesicles by a factor of at least 20. This result suggests that formation of highly curved regions in biological membranes may cause accumulation of certain membrane proteins at those sites.