Asymmetric distribution of GFP-D4H. (A) Single–focal plane differential interference contrast (DIC) and fluorescence micrographs (FMs) of indicated strains expressing GFP-D4H, at indicated temperatures. Arrowheads point to vesicle-like structures. The same micrograph with enhanced brightness is shown. Average ± SEM frequencies of polarized PM staining are indicated. n > 100 cells from three experiments. (B and C) Merged FMs of five 0.1-µm focal planes of cells expressing GFP-D4H and either Sec61-mCherry (B) or Abp1-mCherry (C). Individual channels and merged magnified frames (B) or time lapses (C) are shown. Average ± SEM frequencies of colocalization between cortical GFP-D4H patches and Sec61-mCherry rims (B) or Abp1-mCherry patches (C). n > 100 rims or patches from three experiments. (D) Merged FM of a cell expressing Sec61-YFP, mCherry-D4H, and Abp1-CFP. Individual channels and merged magnified frames are shown. (E) Electron micrographs of a cER rim or ER–endocytic MCSs immunolabeled for HA-GFP-D4H. cER colored in red. Arrowheads point to endocytic invaginations. (F) QEM analysis showing colocalization of HA-GFP-D4H labeling with endocytic proteins and ER rims. Average ± SEM for GRP of the indicated endocytic proteins or the GFP-D4H probe or the ER relative position (ERRP) are indicated. Refer to Fig. S2 A for details. Red asterisks, P < 0.001; orange asterisks, 0.001 ≤ P < 0.1; absence of asterisks, P > 0.1; t test. n > 100 gold particles/invaginations for endocytic proteins and ER rims, and n > 40 for Osh2/3 and GFP-D4H gold particles/invaginations.