FIG 1.
Absence of Fpk1 phosphorylation impairs PtdEth flipping by Dnf1. (A) Strains GFY1770 (DNF1 dnf2Δ dnf3Δ), GFY1773 (DNF1-GFP dnf2Δ dnf3Δ), GFY1772 (DNF16A dnf2Δ dnf3Δ), GFY1775 (DNF16A-GFP dnf2Δ dnf3Δ), and GFY1728 (dnf1Δ dnf2Δ dnf3Δ) were plated as a lawn on yeast extract-peptone-dextrose (YPD) plates, and 10 μl of stock solution of duramycin (either 8 mM or 4 mM) was spotted onto sterile filter paper disks which were immediately placed onto the lawn. Plates were scanned after incubation at 30°C for 2 days. (B) Extracts from GFY1728, GFY1773, and GFY1775 cells were resolved by SDS-PAGE and analyzed by immunoblotting with anti-GFP antibodies. (C) The same cells as in panel B, costained with CellMask Orange to highlight the plasma membrane, were viewed by fluorescence microscopy as described in Materials and Methods.