Fig. 3. The cysteine-rich domain of SNAP-25 is necessary for membrane binding. (A) The SNAP-25A mutants used in this study. BonT/E-resistant SNAP-25 SNAP-25A/ER (S25A/ER) has amino acids ¹⁷⁹Asp and ¹⁸²Met changed to ¹⁷⁹Lys and ¹⁸²Thr. BonT/E-resistant Delta-SNAP-25A/ER (Delta-S25A/ER) lacks 11 amino acids corresponding to the cysteine-rich domain of SNAP-25A and has the corresponding Asp and Met residues changed to Lys and Thr. BonT/E-resistant CA-SNAP-25A/ER (CA-S25A/ER) has all cysteines substituted with alanines and has amino acids ¹⁷⁹Asp and ¹⁸²Met changed to ¹⁷⁹Lys and ¹⁸²Thr. (B) Fractionation of cells transiently expressing wild-type SNAP-25A or Delta-SNAP-25A. Post-nuclear supernatants from these cells were centrifuged as described in Fig. 1 and separated into membrane pellet (P) or cytosol (S). Equal volumes of the fractions were then loaded onto a 13% SDS-PAGE gel, transferred and probed with anti-Myc antibody. (C) Fractionation of cells transiently expressing either wild-type SNAP-25A or CA-SNAP-25A. This experiment was done as described in panel B.