Figure 4.
Enzymatic activity of TAG-synthase sLro1 is required for its recruitment to DAG-enriched ER subdomains. (A) Induction of LD formation by OA results in elevated DAG levels at Fld1 and Nem1 sites. WT cells expressing Fld1-mCherry or Nem1-mCherry and coexpressing the GFP-tagged ER-DAG sensor were diluted and incubated in SC media containing 0.1% OA for 1 h. White arrowheads indicate colocalization of the ER-DAG sensor with Fld1 or Nem1 punctae. Scale bars, 5 µm. (B) Quantification of colocalization between ER-DAG sensor punctae with Fld1 and Nem1 1 h after OA addition. Data are means ± SD, n > 50 cells; ***, P < 0.0005. (C) Cytosolic sLro1 and native Lro1 colocalize with the DAG sensor. 4ΔKO cells expressing mCherry-tagged ER-DAG sensor and coexpressing GFP-FFAT-sLro1/GFP-Lro1 under galactose promoter. White arrowheads denote colocalization between the ER-DAG sensor and either sLro1 or Lro1. Scale bars, 5 µm. (D) Mutant sLro1 fails to produce TAG. 4ΔKO cells expressing WT sLro1 (Scarlet-FFAT-sLro1) or mutant sLro1 (Scarlet-FFAT-sLro1H618A) were grown in galactose media containing [3H]palmitic acid for 21 h. Lipids were extracted and separated by TLC. FFA, free fatty acid. (E) A catalytically inactive mutant of sLro1 fails to produce LDs. 4ΔKO cells expressing WT or mutant version of sLro1 from a galactose-inducible promoter were stained with BODIPY. White arrowheads denote colocalization between BODIPY-marked LDs and WT sLro1 punctae. Scale bars, 5 µm. (F and G) WT and mutant version of sLro1 bind DAG. In vitro binding of the short chain DAG (C8:0) by purified WT and mutant sLro1 was measured by MST. conc, concentration; Fnorm, normalized fluorescence. (H) Enzymatic activity of sLro1 is required for its colocalization with Fld1. 4ΔKO cells expressing WT or mutant version of sLro1 together with Fld1-GFP were switched to galactose media for 2 h. White arrowheads denote colocalization between WT sLro1 and Fld1. Scale bars, 5 µm.

Enzymatic activity of TAG-synthase sLro1 is required for its recruitment to DAG-enriched ER subdomains. (A) Induction of LD formation by OA results in elevated DAG levels at Fld1 and Nem1 sites. WT cells expressing Fld1-mCherry or Nem1-mCherry and coexpressing the GFP-tagged ER-DAG sensor were diluted and incubated in SC media containing 0.1% OA for 1 h. White arrowheads indicate colocalization of the ER-DAG sensor with Fld1 or Nem1 punctae. Scale bars, 5 µm. (B) Quantification of colocalization between ER-DAG sensor punctae with Fld1 and Nem1 1 h after OA addition. Data are means ± SD, n > 50 cells; ***, P < 0.0005. (C) Cytosolic sLro1 and native Lro1 colocalize with the DAG sensor. 4ΔKO cells expressing mCherry-tagged ER-DAG sensor and coexpressing GFP-FFAT-sLro1/GFP-Lro1 under galactose promoter. White arrowheads denote colocalization between the ER-DAG sensor and either sLro1 or Lro1. Scale bars, 5 µm. (D) Mutant sLro1 fails to produce TAG. 4ΔKO cells expressing WT sLro1 (Scarlet-FFAT-sLro1) or mutant sLro1 (Scarlet-FFAT-sLro1H618A) were grown in galactose media containing [3H]palmitic acid for 21 h. Lipids were extracted and separated by TLC. FFA, free fatty acid. (E) A catalytically inactive mutant of sLro1 fails to produce LDs. 4ΔKO cells expressing WT or mutant version of sLro1 from a galactose-inducible promoter were stained with BODIPY. White arrowheads denote colocalization between BODIPY-marked LDs and WT sLro1 punctae. Scale bars, 5 µm. (F and G) WT and mutant version of sLro1 bind DAG. In vitro binding of the short chain DAG (C8:0) by purified WT and mutant sLro1 was measured by MST. conc, concentration; Fnorm, normalized fluorescence. (H) Enzymatic activity of sLro1 is required for its colocalization with Fld1. 4ΔKO cells expressing WT or mutant version of sLro1 together with Fld1-GFP were switched to galactose media for 2 h. White arrowheads denote colocalization between WT sLro1 and Fld1. Scale bars, 5 µm.

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