CHARACTERIZATION OF THE PH-INDUCED FUSION OF LIPOSOMES WITH THE PLASMA-MEMBRANE OF RYE PROTOPLASTS

We present evidence that at acid pH, liposomes composed of soybean lipids fuse with the plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv Puma). Using the resonance energy transfer assay (RET), we 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)phosphatidylethanolamine (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 (t1/2 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.degree. 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. Addition of 2% (v/v) Triton X-100 to these suspensions permitted determination of the total amount of N-NBD-PE associated with the protoplasts. 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. Approximately 64% of the liposomes associated with protoplasts were fused with the protoplasts at pH 3.9, and only 9% at pH 5.6. The transfer of liposome contents to the protoplast interior was studied with 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. Further evidence that the enhancement in calcein fluorescence was not due to release of the dye in the aqueous media came from experiments employing protoplast pellets. After fusion of protoplasts with calcein-containing liposomes, the protoplasts were washed by two centrifugations. Fluorescence intensity measurements and fluorescence microscopy observations showed that protoplasts in the pellets retained a strong calcein fluorescence.