Fig. 1. Amino acids 85–120 of SNAP-25 are sufficient to target GFP to lipid raft domains.

A, residues 85–120 of SNAP-25B, which is the minimal plasma membrane-targeting sequence of this protein. Cysteine residues are highlighted in bold and are underlined. B, transfected PC12 cells were fractionated into cytosol (C) and membrane (M) fractions. The distribution of GFP-SNAP-25 and GFP-85–120 between these fractions was determined by immunoblotting using a GFP-specific antibody. C, Triton X-100-solubilized PC12 cells were fractionated on a discontinuous sucrose gradient as detailed under “Experimental Procedures.” Gradient fractions were analyzed by immunoblotting using antibodies specific for flotillin and transferrin receptor (TfR). Fraction numbers and corresponding sucrose concentrations are indicated; P is the pellet fraction. D, PC12 cells were transfected with GFP-tagged constructs containing full-length SNAP-25B or the membrane-targeting domain (residues 85–120). Purified membranes from transfected cells were solubilized and fractionated on a discontinuous sucrose gradient. Collected fractions were analyzed by immunoblotting using a GFP-specific antibody. E, PC12 cell membranes were incubated in either 1 m hydroxylamine (HA), pH 7, or 1 M Tris, pH 7, at room temperature for 20 h. Membranes were washed, and rafts were purified as Triton-insoluble complexes. Fractions were probed with an antibody specific for SNAP-25.