Fig. S5.

Substitutions at the N220 position can partially suppress drs2Δ cold sensitivity, implying that this position cooperates with acyl-chain selection of substrate. Plasmids carrying Dnf1 or Drs2 were transformed into a drs2∆ genetic background and grown at 30 °C or 20 °C. drs2∆ cells have been shown to exhibit growth defects at 20 °C, which can be suppressed by expressing WT Drs2 or Dnf1^N550S, a Dnf1 mutation that allows the translocation of diacyl-PS across the membrane. The Dnf1^GA-QQ mutation is a gain-of-function substitution that allows lyso-PS transport, but not diacyl-PS, and is unable to suppress the drs2∆ growth inhibition. The Dnf1^N220S and Dnf1^N220R mutations can transport NBD-PS (Fig. 3) and are weakly capable of altering acyl-chain selection. The compound mutants Dnf1^N220S,F1192L and Dnf1^N220S,L1202P appear to suppress the cold sensitivity as does WT Dnf1. The figures presented are representative images taken from three independent transformants examined from three growth experiments.