Figure S1.

Data associated with Fig. 1. TAG-synthase sLro1 needs to be ER associated for its activity. (A and B) sLro1-containing TMDs from Sec63 localize to ER subdomains. Design of the TAG-synthase chimera containing TMDs 1–3 from Sec63 (aa 1-250), facing the active site to the cytoplasm [Sec63(1–250)-GFP-sLro1] (A). 4ΔKO cells expressing Erg6-mCherry and coexpressing Sec63(1–250)-GFP-sLro1 from a galactose-inducible promoter were grown in raffinose media and switched to galactose media for the indicated period of time (B). White arrowheads indicate colocalization of Sec63(1–250)-GFP-sLro1 punctae with Erg6-mCherry. Scale bar, 5 µm. (C) GFP-FFAT-sLro1 localizes to LDs in WT cells. WT cells expressing Erg6-mCherry and coexpressing GFP-FFAT-sLro1 from a galactose-inducible promoter were grown as described in B. White arrowheads indicate colocalization of GFP-FFAT-sLro1 punctae with Erg6-mCherry. The pink arrowhead denotes a GFP-FFAT-sLro1 puncta not associated with Erg6-mCherry. Scale bars, 5 µm. (D–F) The TAG-synthase sLro1 needs to be anchored to the ER membrane for its activity. Design of the soluble fusion protein GFP-sLro1 localized in the cytosol (D), and ssKar2-GFP-sLro1-HDEL localized in the ER lumen (E). 4ΔKO cells expressing Erg6-mCherry and coexpressing either GFP-sLro1 or ssKar2-GFP-sLro1-HDEL from a galactose-inducible promoter. Cells were grown in raffinose media and switched to galactose-containing media for 3 h (F). Scale bars, 5 µm. (G) Nonmembrane-associated sLro1 fails to catalyze TAG synthesis. 4ΔKO cells expressing genomic Erg6-mCherry and coexpressing either native Lro1, GFP-FFAT-sLro1, GFP-sLro1, or ssKar2-GFP-sLro1-HDEL from a galactose-inducible promoter were grown in galactose media containing [³H]palmitic acid for 21 h. Lipids were extracted and separated by TLC. FFA, free fatty acid.