Fig. 5: GRASP55 participates in the Golgi localization and LD targeting of ATGL and MGL.

a Subcellular localization of ATGL and MGL in the intestinal epithelial cells of Grasp55^+/+ and Grasp55^−/− mice was determined using LD floating ultracentrifugation (for Fraction 1), followed by subcellular fractionation assay (for Fractions 2–9; using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g⁻¹ of body weight]) as described in Methods. RAB18/ADRP, Aldolase A, Giantin, ERGIC53, and calreticulin were used as organelle markers of the LDs, cytosol, Golgi, the ER-Golgi intermediate compartment (ERGIC), and the ER, respectively. Immunoblotting results with a full list of proteins involved in lipid metabolism are shown in the Supplementary Fig. 8. The Golgi localizations of ATGL and MGL were reduced by GRASP55 deficiency in mouse intestinal cells (red boxes). Three independent experiments showed similar results. b, c The protein expressions of ATGL, phospho-HSL (pHSL), HSL, MTP, and ADRP were analyzed by immunoblotting. Jejunum tissues were prepared from Grasp55^+/+ and Grasp55^−/− mice fasted for 16 or 4 h after olive oil bolus (olive oil, 10 μl g⁻¹ body weight). Representative immunoblots are shown in (b). Densitometric analysis of ATGL are shown in (c, n = 5). A summary of pHSL, HSL, MTP, and ADRP is shown in Supplementary Fig. 11a–d. The level of β-actin was monitored as a cytosolic protein loading control. d, e Measurements of ATGL in the lipid droplet (LD) fraction of intestinal epithelial cells (using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g⁻¹ of body weight]). Representative immunoblots are shown in (d) and a summary of multiple experiments is shown in (e, n = 3). TM, total membrane. f, g Intracellular localization of ATGL and ADRP, a marker protein of LD, in jejunal epithelia of mice 4 h after olive oil bolus. Representative images are shown in (f) and quantitative analyses of colocalization between ATGL and ADRP are presented in (g, n = 5). MCC, Manders’ colocalization coefficient. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. Scale bars: 20 μm. **p < 0.01. P values were calculated by unpaired (c, g) or paired (e) two-tailed Student’s t tests. Source data are provided as a Source Data file.