Endonexin (annexin IV)-mediated lateral segregation of phosphatidylglycerol in phosphatidylglycerol/phosphatidylcholine membranes

Abstract

Endonexin (annexin IV) is a member of the annexin family of homologous proteins that share the ability to bind to pure lipid membranes and to aggregate vesicles in a Ca(2+)-dependent fashion. Endonexin appears to preferentially interact with certain types of lipids such as phosphatidylglycerol (PG) in PG/phosphatidylcholine (PC) mixed lipid membranes. Such preferential binding should result in localization of PG lipids to membrane regions where endonexin is bound. This was tested using a PG derivative containing the fluorophore pyrene, which exhibits fluorescence sensitive to molecular collision frequency. Motional restriction of pyrene-PG upon endonexin-membrane binding was evident from decreased ratios of excimer-to-monomer (E/M) pyrene fluorescence with endonexin binding to 97% PG/3% pyrene-PG vesicles. A maximum decrease of 30% suggests a 30% decrease in the average diffusion constant of pyrene-PG molecules or a 53% decrease assuming that only outer-monolayer lipid molecules interact with endonexin. In vesicles containing 5% and 10% pyrene-PG in PC, segregation of lipids was evident from observed increases in E/M of 14.2 +/- 1.8% and 6.8 +/- 0.1%, respectively, in the presence of endonexin and either 10 mM (5% pyrene-PG) or 2 mM (10% pyrene-PG) free Ca2+. At higher concentrations of Ca2+ (> 10 mM for 5% pyrene-PG and > 2 mM for 10% pyrene-PG), smaller endonexin-dependent increases in E/M are observed as endonexin molecules at high surface densities compete for the limited pool of pyrene-PG. The nature of these interactions of endonexin with mixed lipid systems has implications for the way annexins may modulate membrane structure in cells.

Two tail-anchored protein variants, differing in transmembrane domain length and intracellular sorting, interact differently with lipids

“…To confirm the different interaction of the two b 5 forms with lipid domains, we applied an alternative method, based on fluorescence measurements of excimer formation coupled to the use of the pyrene-labeled fluorescent probes pyrene-PS or pyrene-PC inserted in POPC liposomes. Changes in excimer͞monomer intensity ratios induced by associated proteins can give information on the interactions of the proteins with the lipid probes or with the lipid domains that contain them (23).…”

A New Consensus Sequence for Phosphatidylserine Recognition by Annexins

“…These secondary DE calcium-binding sites could play a functional role, however, once the protein is bound to the membrane interface by transiently binding calcium and phospholipids and thus could contribute to reduce the global lateral diffusion of these two species (26,27). The consensus sequence for the equatorial PS-binding site was found exclusively in domain 1 or domain 2 but not in the other two domains.…”

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

“…This was unexpected based on earlier analyses, which have generally implied that the net entropy change should be negative for several reasons. Multiple studies have shown that membrane phospholipids are clustered (38,39) and/or immobilized (40, 41) upon binding of annexins, and the membrane becomes more rigid (42), all factors that should cause a reduction in entropy. Likewise, the formation of ordered multimeric complexes of annexin V on the vesicle surface (13, 18 -21) should also reduce entropy.…”