Absence of Fld1 from LD biogenesis sites results in ectopic droplet formation. (A and B) LDs form at Fld1- and Nem1-marked ER sites. 3ΔKO expressing Fld1-mCherry (A) or Nem1-mCherry (B) and coexpressing Erg6-GFP were transferred to galactose-containing media for the indicated time. White arrowheads denote colocalization between Erg6 with Fld1 (A) or Nem1 (B). (C) Quantification of colocalization of Erg6 with Fld1 and Nem1. Data are means ± SD, n > 50 cells. (D) LDs fail to form at Nem1 sites in cells lacking Fld1. fld1Δ mutant cells (fld1Δ 3ΔKO) expressing Nem1-mCherry and coexpressing Erg6-GFP were grown as described in A. White arrowheads indicate Erg6 punctae that do not colocalize with Nem1. Scale bar, 5 µm. (E and F) Quantification of cells with at least 1 LD (E), or number of LDs per cell (F) for images shown in D. Data are means ± SD, n > 50 cells; ***, P < 0.0005. (G) Cartoon depicting lack of Fld1 from LD biogenesis sites does not affect the localization of Nem1 and Yft2 but results in mislocalization of Pex30 and failure to recruit the TAG-synthase. (H) Cartoon showing that ER subdomains containing Fld1, Nem1, Yft2, and Pex30 define sites for the localization of the TAG-synthase and droplet formation. Erg6 and Pet10 bind to newly formed LDs from the cytosolic side.