Identification of motifs and mechanisms for lipid droplet targeting of the lipolytic inhibitors G0S2 and HIG2

Abstract

G0S2 and HIG2 are two selective inhibitors of ATGL (also known as PNPLA2), the key enzyme for intracellular lipolysis. Whereas G0S2 regulates triglyceride (TG) mobilization in adipocytes and hepatocytes, HIG2 functions to enhance intracellular TG accumulation under hypoxic conditions. A homologous hydrophobic domain (HD) is shared by G0S2 and HIG2 (also known as HILPDA) for binding to ATGL. However, the determinants of their lipid droplet (LD) localization are unknown. Here, we study how G0S2 and HIG2 are targeted to LDs, and identify both ATGL-independent and -dependent mechanisms. Structural prediction and studies in cells reveal that ATGL-independent localization of G0S2 to both the endoplasmic reticulum (ER) and LDs is mediated by a hairpin structure consisting of two hydrophobic sequences. Positively charged residues in the hinge region play a crucial role in sorting G0S2, which initially localizes to ER, to LDs. Interestingly, the role of these positive charges becomes dispensable when ATGL is co-expressed. In comparison, HIG2, which lacks a similar hairpin structure, is dependent on ATGL for its full LD targeting. Thus, our studies identify specific structural features and mechanisms for mediating accumulation of these two ATGL inhibitors on LDs.

Keywords: Endoplasmic reticulum; Inhibitor; Lipase; Lipid droplet; Lipolysis; Localization; Motif.

Fig. 1.

Localization of G0S2 in mouse primary hepatocytes. Lipid-starved primary ATGL^−/− hepatocytes expressing either G0S2–FLAG alone (A) or with Myc–ATGL (WT or S47A mutant) (C) were treated with or without oleic acid (OA) overnight, and then imaged by immunofluorescence confocal microscopy. LDs were stained with BODIPY. Scale bars: 20 µm. The white box indicates the enlarged areas. (B,D) Mean±s.d. (n=20) of the protein signal in A and C on LDs are shown in B and D, respectively. ***P<0.001; NS, not significant (unpaired two-tailed t-test).

Fig. 2.

Distribution of G0S2 in ER and LDs upon LD formation. Lipid starved ATGL^−/− HeLa cells expressing G0S2–FLAG and GFP–Sec61 were treated with oleic acid for various lengths of time, and then imaged by immunofluorescence confocal microscopy. Scale bars: 10 µm. The white box indicates the enlarged areas. Images representative of three independent repeats.

Fig. 3.

The hairpin structure targets G0S2 to ER. (A) The amino acid sequences of human and mouse G0S2 were aligned using Clustal Omega program. Overall, the sequences are well conserved, with high levels of basic (shown in green) and hydrophobic (shown in blue) residues. Red boxes contain the hydrophobic helical sequences. Identical amino acids in all proteins are marked with an asterisk (*), conservative substitutions with a colon (:), and semi conservative substitutions with a period (.). (B) HeLa cells expressing WT or deletion mutants of G0S2 along with GFG–Sec61 were treated with oleic acid and then imaged by immunofluorescence confocal microscopy. Scale bars: 10 µm. (C) The colocalization efficiency (mean±s.d.) between the G0S2 and GFP–Sec61 is shown. ***P<0.001 (unpaired two-tailed t-test). The Pearson's correlation coefficient was employed to quantitate the colocalization efficiency. Three repeats of each treatment were processed with ImageJ to obtain Pearson's correlation coefficient.

Fig. 4.

Positively charged residues in the hinge region sort G0S2 from ER to LDs. (A) HeLa cells expressing WT or point mutants of G0S2 were treated with oleic acid and then imaged by immunofluorescence confocal microscopy. LDs were stained with BODIPY. (B) Mean±s.d. (n=20) of the protein signal on LDs are shown. ***P<0.001; NS: no significance (unpaired two-tailed t-test). (C) HeLa cells expressing G0S2/R20A/K22A/K25A triple mutant and GFP–Sec61 were treated with oleic acid and then imaged by immunofluorescence confocal microscopy. Images representative of three independent repeats. Scale bars: 10 µm. The white boxes indicates the enlarged areas.

Fig. 5.

The motif of G0S2 is required for ATGL interaction but for LD localization. (A) Schematic showing the structures of engineered deletion mutants of G0S2. The location of the HD domain is indicated by gray bars starting from residues 27 to 42. The HD domain was divided to 4 parts, 27–30 (1), 31–34 (2), 35–38 (3), and 39–42 (4), respectively, and the corresponding mutants were constructed with a C-terminal FLAG-tag. (B) FLAG-tagged mutants were co-expressed with Myc–ATGL in HeLa cells and analyzed by anti-FLAG immunoprecipitation (IP) followed by immunoblotting. Input represents 5%. (C) HeLa cells expressing WT or deletion mutants of G0S2 were treated with oleic acid and then imaged by immunofluorescence confocal microscopy. LDs were stained with BODIPY. Scale bars: 10 µm. Images in B and C representative of three independent repeats.

Fig. 6.

ATGL coexpression promotes LD localization of G0S2 independently of positively charged residues. (A) In vitro translated ATGL was mixed with WT or R20A/K22A/K25A mutant of G0S2 and TG hydrolase activity was measured using [³H] triolein as substrate. The data are representative of three independent experiments (mean±s.d.). *P<0.05, **P<0.01 versus vector (unpaired two-tailed t-test). (B,C) HeLa cells expressing G0S2/R20A/K22A/K25A or G0S2/R20A/K22A/K25A/Δ27-30 mutant with WT ATGL or ATGLΔLBD mutant were treated with oleic acid and then imaged by immunofluorescence confocal microscopy. LDs were stained with LipidTOX dye. Scale bars: 10 µm.

Fig. 7.

Distribution of HIG2 in ER and LDs upon LD formation. (A) Lipid-starved ATGL^−/− HeLa cells expressing HIG2–FLAG and GFP–Sec61 were treated with oleic acid for various lengths of time. (B) ATGL^−/− HeLa cells expressing HIG2–FLAG with WT ATGL, ATGL/S47A, or ATGLΔLBD mutant were treated with oleic acid for 15 h. (C) ATGL^−/− HeLa cells expressing G0S2/HIG2 hybrid proteins were treated with oleic acid for 15 h. G0S2/HIG2 fusion1 contains the hinge sequence of G0S2 fused to the N-terminus of full-length HIG2. G0S2/HIG2 fusion2 contains the first helix and hinge sequence of G0S2 fused to the N terminus of full-length HIG2. All cells were imaged by immunofluorescence confocal microscopy. LDs were stained with LipidTOX dye. Scale bars: 10 µm. Images representative of three independent repeats.