Extended Data Fig. 5: Membrane-fusion regulator, tether, and SNAREs are required for late ER-to-LD targeting.
From: Identification of two pathways mediating protein targeting from ER to lipid droplets
a, Western blot analysis of fractions of wildtype cells upon RNAi and LD induction. GPAT4 amounts remain the same or increase in membrane fractions but decrease in LD fractions upon depletion of membrane-fusion machinery components. Left: protein targets; right: ladder positions. I = Input (whole-cell lysate), S = soluble fraction. GPAT4 band intensities in membrane fractions: LacZ (1.00), Trs20 (2.25 ± 0.58), Rab1* (2.62 ± 1.04), Rint1 (2.11 ± 0.25), Syx5** (3.07 ± 0.40), and Bet1 (2.17 ± 0.28) (mean ± SD, n = 3). One-way ANOVA with Bonferroni correction, *p = 0.0455, **p = 0.0074, compared to LacZ. b, HSD17B11 requires membrane-fusion machinery components for LD targeting. Confocal imaging of live wildtype cells transiently transfected with HSD17B11-EGFP upon RNAi of fusion machinery, followed by a 20-hr incubation in oleate-containing medium. Scale bar, 5 and 1 μm (inlay). c, Bar graph showing percentage of cells with LD targeting (defined as those with >2 LDs with protein targeting) from the imaging experiment in b. Mean ± SD, n = 6 experiments for LacZ RNAi and 3 experiments for the rest (12–17 cells each). One-way ANOVA with Bonferroni correction, #p < 0.0001, compared to LacZ. d, e, Bar graph showing percentage of cells for a given number of LDs with HSD17B11-EGFP targeting upon RNAi of membrane-fusion machinery. Representative images in b. f, Depletion of specific Rab, membrane-tethering complex components, and SNAREs does not impair targeting of Ubxd8 (early ER-to-LD targeting) or CGI-58 and CCT1 (cytosol-to-LD targeting). Confocal imaging of live wildtype cells transiently transfected with EGFP- or mCherry-tagged constructs upon RNAi of fusion machinery, followed by a 20-hr incubation in oleate-containing medium. Scale bar, 5 and 1 μm (inlay). g, Bar graph showing targeting ratios from the imaging experiment in f. Mean ± SD, n = (left to right; top to bottom) 85, 35, 34, 36, 36, 34; 74, 33, 33, 31, 33, 31; 54, 36, 32, 33, 29, 32 cells examined over 3 independent experiments. For mCherry-CCT1, nuclear signal was excluded from the calculation. One-way ANOVA with Bonferroni correction, #p < 0.0001, compared to LacZ. Source numerical data and unprocessed blots are available in source data.
