Fld1 and Nem1 are both required for recruitment of the TAG-synthase. (A and B) Fld1 is required to recruit both native and cytosolic Lro1 to Nem1 sites. fld1Δ cells expressing Nem1-mCherry and coexpressing native GFP-Lro1 (A) or cytosolic GFP-FFAT-sLro1 (B) from a galactose-inducible promoter. Scale bars, 5 µm. (C and D) Nem1 is required to recruit Lro1 to Fld1 sites. nem1Δ cells expressing Fld1-mCherry and coexpressing native GFP-Lro1 (C) or cytosolic GFP-FFAT-sLro1 (D) from a galactose-inducible promoter. Scale bars, 5 µm. (E and F) Fld1 and Nem1 together constitute functional ER sites for LD biogenesis. Cartoon depicting ER subdomains containing both Fld1 and Nem1 as a prerequisite for the recruitment of the native GFP-Lro1 or the cytosolic GFP-FFAT-sLro1. (G) Expression of Pah1-7P in nem1Δ cells rescues the defect in recruitment of the TAG-synthase. nem1Δ cells expressing Fld1-mCherry, and coexpressing both Pah1-7P, and GFP-FFAT-sLro1. White arrowheads denote colocalization between GFP-FFAT-sLro1 and Fld1-mCherry. Scale bar, 5 µm.