Fig. 4.

N220 cooperates with L1202 of TM5 to increase SM selection and preference. (A) Homology model of Dnf1 (3W5D) with TM helices 1–6 shown as pink cylinders and numbered, with key residues represented in stick form and colored by element. Dashed blue lines illustrate the exit gate triad of N220, N550, and Y618; these residues participate in substrate backbone, acyl chain, and headgroup selection, respectively. The rotated view of the model reveals alternative orientation of PL substrate within the exit gate, consistent with the positioning of headgroup-selective, backbone-selective, and acyl chain-selective residues as indicated and shown in sticks. PE is shown in the model as sticks with shaded spheres to illustrate spacing and colored by element. (B) Comparisons of PC, PE, PS, and SM transport among I615M, I615S, I615T, and N220S compound alleles with preferences for SM (C) and PS (D). n ≥ 9 ± SEM for all 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. (E) An alignment of P4-ATPase sequences reveals conservation of F1192 but not L1202. (F) Cartoon schematic of TM-domain substrate transport indicating the putative secondary influence of L1202 for PS selection at the entry gate and the primary selective role of N220 for SM selection at the exit gate.