Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one perilipin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific perilipins present on its LDs. Despite their early discovery, exactly how perilipins target LDs and why they displace each other in a ‘hierarchical’ manner remains unclear. They all share an amino-terminal 11-mer repeat amphipathic region suggested to be involved in LD targeting. Here, we show that in vivo this domain functions as a primary highly reversible LD targeting motif in perilipins1-3 and, in vitro, we document reversible and competitive binding between a wildtype purified perilipin1 11-mer repeat peptide and a mutant with reduced binding affinity to both ‘naked’ and phospholipid coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in perilipin1 more effectively than in perilipin2, whereas in perilipin3 it weakens binding. These findings suggest that dual amphipathic helical regions mediat
