The phospholipid monolayer associated with perilipin-enriched lipid droplets is a highly organized rigid membrane structure.

The significance of lipid droplets (LD) in lipid metabolism, cell signaling, and membrane trafficking is increasingly recognized, yet the role of the LD phospholipid monolayer in LD protein targeting and function remains unknown. To begin to address this issue, two populations of LD were isolated by ConA sepharose affinity chromatography: 1) functionally active LD enriched in perilipin, caveolin-1, and several lipolytic proteins, including ATGL and HSL; and 2) LD enriched in ADRP and TIP47 that contained little to no lipase activity. Coimmunoprecipitation experiments confirmed the close association of caveolin and perilipin and lack of interaction between caveolin and ADRP, in keeping with the separation observed with the ConA procedure. The phospholipid monolayer structure was evaluated to reveal that the perilipin-enriched LD exhibited increased rigidity (less fluidity), as shown by increased cholesterol/phospholipid, Sat/Unsat, and Sat/MUFA ratios. These results were confirmed by DPH-TMA, NBD-cholesterol, and NBD-sphingomyelin fluorescence polarization studies. By structure and organization, the perilipin-enriched LD most closely resembled the adipocyte PM. In contrast, the ADRP/TIP47-enriched LD contained a more fluid monolayer membrane, reflecting decreased polarizations and lipid order based on phospholipid fatty acid analysis. Taken together, results indicate that perilipin and associated lipolytic enzymes target areas in the phospholipid monolayer that are highly organized and rigid, similar in structure to localized areas of the PM where cholesterol and fatty acid uptake and efflux occur.