FIGURE 2.

Dnf1 suppressors of drs2Δ cold-sensitive growth transport NBD-PS across the plasma membrane and restore endogenous PS asymmetry. A, growth of drs2Δ expressing the indicated Drs2 and Dnf1 variants at 30 °C or 20 °C. B, dnf1Δdnf2Δ cells expressing the indicated Dnf1 variants were incubated with NBD-phospholipid for 30 min at 4 °C as described under “Experimental Procedures.” The amount of lipid transported was normalized to NBD-PC uptake by WT Dnf1. C, the data shown in B was graphed as the ratio of NBD-PE to NBD-PC uptake (open bars) or NBD-PS to NBD-PC uptake (gray bars) to assess relative changes in substrate preference. The dotted lines are the ratios for WT Dnf1, and the shaded region above and below these lines are confidence intervals. Values extending outside of the confidence intervals represent a significant change in substrate specificity. D, the Dnf1 variants indicated were tested for their ability to restore PapB resistance (PS asymmetry) to a drs2Δ strain. The Dnf1 suppressors of drs2Δ cold-sensitive growth restore PS asymmetry better than WT Dnf1.