FIGURE 2.

Cys⁵⁰ of SMS1 and Cys³⁴³/Cys³⁴⁸ of SMS2 are involved in cross-linking by cysteine cross-linking reagents. A–F, COS7 cells were transfected with a plasmid encoding FLAG-tagged SMS1-WT or SMS1 with endogenous cysteine substitutions (C25S, C50S, C145S, C187S, C217S, C227S, C244S, C277S, C311S, or C321S) (A, C, and E) or SMS2-WT or SMS2 with endogenous cysteine substitutions (C160S, C171S, C188S, C221S, C255S, C265S, C331S and C332S, or C343S and C348S) (B, D, and F). 24 h post-transfection, the cells were treated with 20 μm BMH (spacer arm, 13.0 Å) (A and B) or 20 μm of BMB (spacer arm, 10.9 Å) or BMOE (spacer arm, 8.0 Å) (C and D) for 15 min. A–D, the cells were lysed and analyzed by immunoblotting with anti-FLAG antibody. Results are from one experiment representative of three independent experiments. E and F, SMS activity in the lysate of COS7 cells expressing WT or cysteine-substituted SMSs were determined using C6-NBD-Cer as a substrate. Reaction mixtures containing cell lysates (20 μg of protein/assay) were incubated at 37 °C for 30 min. Individual data points are shown as a scatterplot. Values represent the mean ± S.D. from three independent experiments. *, p < 0.01; **, p < 0.05.