Figure 1.
The TAG-synthase Lro1 reveals sites of nascent LD biogenesis in the ER. (A) Topology of TAG-synthase Lro1. N, nucleus. (B) Design of the MBP-GFP-FFAT-sLro1 chimera, which binds the ER-resident Scs2 through a FFAT motif and has its catalytic domain exposed to the cytoplasm. (C and D) GFP-FFAT-sLro1 is enzymatically active. Cells of indicated genotypes were grown in SC raffinose media, diluted in SC galactose containing [3H]palmitic acid, and grown for the indicated period of time. Lipids were extracted and separated by TLC. TAG-to-phospholipids ratio is plotted in C, and TLC image of 21-h time point is shown in D. Data represent mean ± SD of three independent experiments. (E and F) GFP-FFAT-sLro1 localizes to discrete ER subdomains. 4ΔKO cells expressing genomic Erg6-mCherry and coexpressing GFP-FFAT-sLro1 (E) or GFP-FFAT (F) from a galactose-inducible promoter. Arrowheads indicate colocalization between GFP-FFAT-sLro1 and Erg6-mCherry. Scale bar, 5 µm. (G) Western blot showing expression of GFP-FFAT-sLro1 or Kar2 after galactose induction for the indicated period of time. (H) Quantification of colocalization between GFP-FFAT-sLro1 and Erg6-mCherry. Percentage of GFP-FFAT-sLro1 puncta that colocalize with Erg6-mCherry after galactose induction. Data represent mean ± SD, n > 50 cells; ***, P < 0.0005. (I) GFP-FFAT-sLro1 colocalizes with LDs. CLEM imaging of 4ΔKO cells expressing GFP-FFAT-sLro1. Cells were induced in galactose for 1 h. GFP-FFAT-sLro1 is enriched at a discrete site in the ER by fluorescence imaging and colocalizes with an electron-translucent LD by EM. Yellow arrow demarcates ER, white arrow denotes GFP-FFAT-sLro1 puncta, and black arrow denotes an LD. N, nucleus.

The TAG-synthase Lro1 reveals sites of nascent LD biogenesis in the ER. (A) Topology of TAG-synthase Lro1. N, nucleus. (B) Design of the MBP-GFP-FFAT-sLro1 chimera, which binds the ER-resident Scs2 through a FFAT motif and has its catalytic domain exposed to the cytoplasm. (C and D) GFP-FFAT-sLro1 is enzymatically active. Cells of indicated genotypes were grown in SC raffinose media, diluted in SC galactose containing [3H]palmitic acid, and grown for the indicated period of time. Lipids were extracted and separated by TLC. TAG-to-phospholipids ratio is plotted in C, and TLC image of 21-h time point is shown in D. Data represent mean ± SD of three independent experiments. (E and F) GFP-FFAT-sLro1 localizes to discrete ER subdomains. 4ΔKO cells expressing genomic Erg6-mCherry and coexpressing GFP-FFAT-sLro1 (E) or GFP-FFAT (F) from a galactose-inducible promoter. Arrowheads indicate colocalization between GFP-FFAT-sLro1 and Erg6-mCherry. Scale bar, 5 µm. (G) Western blot showing expression of GFP-FFAT-sLro1 or Kar2 after galactose induction for the indicated period of time. (H) Quantification of colocalization between GFP-FFAT-sLro1 and Erg6-mCherry. Percentage of GFP-FFAT-sLro1 puncta that colocalize with Erg6-mCherry after galactose induction. Data represent mean ± SD, n > 50 cells; ***, P < 0.0005. (I) GFP-FFAT-sLro1 colocalizes with LDs. CLEM imaging of 4ΔKO cells expressing GFP-FFAT-sLro1. Cells were induced in galactose for 1 h. GFP-FFAT-sLro1 is enriched at a discrete site in the ER by fluorescence imaging and colocalizes with an electron-translucent LD by EM. Yellow arrow demarcates ER, white arrow denotes GFP-FFAT-sLro1 puncta, and black arrow denotes an LD. N, nucleus.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal