Fig. 2.

Hydroxyl, but not sulfhydryl, substitutions at the highly conserved N220 are sufficient for SM translocation. (A and B) A serine at position N220 increases NBD-SM selection (A) and preference (B). (C) An alignment of diverse P4-ATPase sequences from mammals to fungi indicates conservation of N220. (D) Substrate transport measurements of alternative biochemical substitutions at position 220 demonstrate the coordination of backbone and headgroup selection. n ≥ 3 for PA examinations, and n ≥ 9 ± SEM for all remaining data. Comparisons to WT ratio or substrate uptake were made with one-way ANOVA using Tukey’s post hoc analysis; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.