RUNNING TITLESProtein targeting to adiposomes and enzymatic activity. ABBREVIATIONS DOPC: 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine. DOPE: 1,2-di-(9Z-octadecenoyl)-sn-glycero-3phosphoethanolamine. PI: phosphatidylinositol. Liver PI: L-α-phosphatidylinositol (Liver, Bovine) (sodium salt). TAG: triacylglycerol. ADRP: adipose differentiation-related protein. TIP47: tail-interacting protein of 47 kDa. PLINs: perilipin family proteins. ATGL: adipose triglyceride lipase. ALD: artificial lipid droplet. LD: lipid droplet. FFF-MALS: 2 field flow fractionation-multi-angle light scattering. TEM: Transmission electron microscope. SUMMARYNew strategies to decode the specific protein targeting mechanism on lipid droplet (LD) are urgently needed.Using adiposome, the LD binding of perilipin 2 (PLIN2), perilipin 3 (PLIN3), and adipose triglyceride lipase (ATGL) were studied. Scatchard analysis found that the binding of PLIN2 to the adiposome surface was saturable, pointing to a specific membrane binding partner. Phosphatidylinositol (PI) was found to inhibit PLIN2 binding while it did not impede PLIN3. Structural analysis combined with mutagenesis revealed that the 73 rd glutamic acid of PLIN2 is significant for the effect of PI on the protein binding. The presence of PI significantly stimulated the activity of ATGL in vitro. The phosphorylation site mutants of ATGL were found reducing the lipase activity in the adiposome system. Our study demonstrates the utility of adiposome as a powerful, manipulatable model system for the characterization of LD binding and enzymatic activity of LD proteins in vitro.