Bovine annexin IV (endonexin) was bound to supported planar bilayers composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) in the first monolayer facing the substrate, and varying mole fractions of POPC, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and small amounts of the fluorescent lipid analogs NBD-PC or NBD-PG in the second monolayer facing the large aqueous compartment. Lateral diffusion coefficients and mobile fractions of these phospholipids were measured by fluorescence recovery after photobleaching (FRAP) as a function of protein concentration and lipid composition in the presence of 2 mM CaCl2 or 1 mM EDTA. In the absence of annexin IV, the lateral diffusion coefficients depended only little on the POPC:POPC ratios and were approximately 3.0 mu m(2)/s for NBD-PG (no Ca2+), 2.5 mu m(2)/s for NBD-PG (2 mM Ca2+), and 1.6 mu m(2)/s for NBD-PC (with or without 2 mM Ca2+). In the presence of 2 mM Ca2+ these diffusion coefficients decreased as a function of the added annexin concentration. A transition from a state with ''rapid'' lipid diffusion to a state with ''slow'' lipid diffusion occurred at about 80 nM annexin IV and was independent of the POPC:POPG ratio. In addition to reducing the lipid lateral diffusion coefficients, annexin IV also gave rise to two-component lateral diffusion of the lipids in these mixed bilayers. The split of the single diffusion coefficient of NBD-PG into two components occurred at most POPC:POPG ratios upon binding of annexin IV, but required higher annexin concentrations at mole fractions of POPC between 66 and 82 mol % than at high mole fractions of POPG or 90 mol % POPC. The magnitude of the decrease of both lateral diffusion coefficients depended strongly on the amount of POPG in the membrane, ranging from a 0.8- and 1.6-fold decrease at 10 mol % POPG to a 5- and 35-fold decrease for the higher and lower diffusion coefficient, respectively, at 99 mol % POPG. Since similar results were obtained with the NBD-PG and NBD-PC lipid probes, it is concluded that in the presence of Ca2+ annexin IV induces a fluid-fluid phase separation in these membranes in a POPG-dependent fashion rather than forming long-lived stoichiometric complexes with the POPG molecules.
