Specificity for the exchange of phospholipids through polymyxin B mediated intermembrane molecular contacts

Abstract

Structural specificity for the direct vesicle-vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (P×B) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299-308], intermembrane molecular contacts between anionic vesicles are formed by a small number of P×B molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of P×B to vesicles, formation of stable vesicle-vesicle contacts, and exchange of phospholipids. Polycationic P×B binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through P×B contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. P×B does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by P×B. Vesicles of dianionic phospholipids, like phosphatidic acid, bind P×B; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of P×B at the interface. P×B in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that P×B at the interface can exist in two forms depending on the lipid/P×B ratio. Additional studies show that stable P×B-mediated vesicle-vesicle contacts may be structurally and functionally distinct from "stalks", the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.