Fig. S1.

The FACS SM⁺ screen identifies substitutions through TM1, -2, -3, -4, and -5 that are capable of increasing NBD-SM transport. (A) Dnf1 mutant libraries were gated to select viable, single-cell populations for sorting. Cells containing the top ∼1% of the FITC signal were sorted and cultured. (B) The first-generation hits from the FACS SM⁺ screen reveal several alleles capable of translocating SM. Several of the SM⁺ alleles bore substitutions at the same position (e.g., L1202P and L1202S), whereas others were characterized by identical substitutions from different codon changes (e.g., C564S); these observations support previous assertions regarding the complexity of this mutagenesis library (39). n ≥ 9, ± SEM; the asterisk indicates single or double Dnf1 mutants that doubled SM preference relative to WT (see Fig. 1C).