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

G0S2 and HIG2 are two selective inhibitors of ATGL, the key enzyme for intracellular lipolysis. While 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 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 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 at 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.