The ER lipid phosphatase Sac1 is closely apposed to the PM. (A) Inverted (negative contrast), maximum-intensity projection of a pair of tsA201 cells expressing mCherry-Sec61β ER marker. Inset is 3× magnification. (B) Inverted (negative contrast), maximum-intensity projection of a tsA201 cell expressing GFP-Sac1. Inset is 2× magnification. (C) Superresolution_EPI localization map of a region of interest from a fixed tsA201 cell stained for anti-mCherry against mCherry-Sec61β (left), anti-Sac1 (middle), and merge (right). (D) Inverted TIRF footprint of a tsA201 cell expressing GFP-Sac1. (E) Inverted TIRF footprint from a tsA201 cell fixed and stained for anti-Sac1. (F) Superresolution_TIRF localization map from a fixed tsA201 cell expressing GFP-Sac1. (G, top) TIRF footprint from a single tsA201 cell expressing MAPPER (left), Sac1 (middle), and the region of the TIRF footprint with overlapping signals (Sac1–PM; right). Inset is 1.5× magnification. (bottom) Representative intensity profile of a region of the TIRF footprint. Note that the signals are coincident with one another. (H, top) Confocal micrograph of a single tsA201 cell expressing GFP-Sac1 (donor); middle: same cell, expressing RFP-LDR (acceptor); right: FRET signal. (middle) Representative profile plot taken between the arrows of the FRET image in the top panel. (H, bottom) Quantification of N_FRET (means ± SEM, n = 8–11; t test; *, P < 0.05; **, P < 0.01).