Fig. 1.

The FACS SM⁺ screen identifies substitutions throughout TM1, -2, -3, and -5 of Dnf1 that are capable of increasing NBD-SM transport. (A) The first six TM segments were mutagenized by error-prone PCR and selected through FACS for their ability to transport NBD-SM. (B) NBD-SM differs from NBD-PC only in its sphingosine backbone. (C) Five substitutions that double the preference of Dnf1 for NBD-SM were isolated. (D) Measurements of alternative NBD-labeled PLs demonstrate substrate specificity in the SM⁺ alleles. Negative values occur when the uptake of vector-only control NBD-PL is greater than the uptake of experimental samples. 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 < 0.001; ****P < 0.0001.