Characterization of the pH-induced fusion of liposomes with the plasma membrane of rye protoplasts

Abstract

The authors present evidence that at acidic pH, liposomes composed of soybean lipids fuse with the plasma membrane of protoplasts isolated from rye leaves. Using the resonance energy transfer assay (RET), they determined the rate and extent of liposome and protoplast plasma membrane lipid mixing. The fluorescent donor-acceptor pair was N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidyl-ethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE). Fusion was substantial below pH 5, and the half-time of lipid mixing was fast (t/sub 1/2/ on the order of minutes) and pH, concentration, and temperature dependent. The extent of liposome and protoplast fusion from the total amount of liposomes associated with the protoplasts was also determined by the RET assay. Protoplasts were incubated with fluorescent-labeled liposomes (5 min at 30/sup 0/C) at different pH values and then washed twice by centrifugation. The fluorescence spectra of the protoplast suspension permitted determination of the ratio of N-NBD-PE emission at 530 nm to the N-Rh-PE emission at 590 nm, which is a measure of the degree of lipid mixing. The amount of liposomes associated (fused and unfused) with protoplasts at pH 3.9 was approximately 9 times greater than that at pH 5.6. The transfer of liposome contents to the protoplast interior was studied withmore » a method based on the fluorescence enhancement of a solution of calcein, initially confined in the liposomes at self-quenching concentrations. The kinetics of calcein release were very similar to those of lipid mixing. Fluorescence microscopy showed that after fusion with liposomes containing calcein, the protoplasts exhibited a strong diffuse fluorescence in the interior.« less