Interactions between lipid-anchored and transmembrane proteins. Spin-labelESR studies on avidin-biotinyl phosphatidylethanolamine in membrane recombinants with myelin proteolipid proteins

Interactions between lipid-anchored and transmembrane proteins are relevant to the intracellular membrane sorting of glycosyl phosphatidylinositol-lin ked proteins. We have studied the interaction of a spin-labeled biotinyl di acyl phospholipid, with and without specifically bound avidin, with the mye lin proteolipid protein (or the DM-20 isoform) reconstituted in dimyristoyl phosphatidylcholine, Tetrameric avidin bound to the N-biotinyl lipid headgr oup is a surface-anchored protein, and the myelin proteolipid is an integra l protein containing four transmembrane helices. The electron spin resonanc e (ESR) spectrum of N-biotinyl phosphatidylethanolamine spin-labeled at the C-14 position of the sn-2 chain consists of two components in fluid-phase membranes of dimyristoylphosphatidylcholine containing the proteolipid, In the absence of avidin, this is characteristic of lipid-protein interactions with integral transmembrane proteins. The more motionally restricted compo nent represents the lipid population in direct contact with the intramembra nous surface of the integral protein, and the more mobile component corresp onds to the bulk fluid lipid environment of the bilayer. In the presence of avidin, the biotin-lipid chains have reduced mobility because of the bindi ng to avidin, even in the absence of the proteolipid [Swamy, M. J., and Mar sh, D, (1997) Biochemistry 36, 7403-7407], In the presence of the proteolip id, the major fraction of the avidin-anchored chains is further restricted in its mobility by interaction with the transmembrane protein. At a biotin- lipid concentration of 1 mol %, approximately 80% of the avidin-linked chai ns are restricted in membranes with a phosphatidylcholine: proteolipid mola r ratio of 37:1. This relatively high stoichiometry of interaction can be e xplained when allowance is made for the closest interaction distance betwee n the lipid-anchored avidin tetramer and the transmembrane proteolipid hexa mer, without any specific interaction between the two types of membrane-ass ociated proteins, The interaction is essentially one of steric exclusion, b ut the lipid chains are rendered more sensitive to interaction with the int egral protein by being linked to avidin, even though they are removed from the immediate intramembrane proteinlipid interface. This could have implica tions for the tendency of lipid-anchored chains to associate with membrane domains with reduced lipid mobility.