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

Abstract

(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, NBDcholesterol, 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. lipolysis; caveolae; caveolin; concanavalin-A; adipose differentiationrelated protein IT IS INCREASINGLY CLEAR THAT LIPID DROPLETS (LD) are dynamic organelles with regulatory roles in many cellular processes besides lipid metabolism, including cell signaling, immune function, membrane trafficking, and regulation of longevity (42,70). Whereas the mechanism of these processes is only partially understood, much less is known about how the structure of the LD phospholipid monolayer may affect LD protein targeting and function. Embedded in the monolayer that surrounds the LD neutral lipid (NL) core are proteins, such as perilipins (65) and adipose differentiation-related protein (ADRP) (16,41), that coat the LD surface and lipids such as cholesterol (4, 80) that help to define the LD structure. Perilipin and ADRP are two members of the PAT [perilipin, ADRP, tail-interacting protein 47 (TIP47)] family from the Plin group of genes (56) that were initially thought to act simply as barriers to protect the NL core against lipolytic action (63, 65). Current evidence suggests a more complex mechanism, one that requires coordination of perilipin with several lipases, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the activator molecule comparative gene identification-58 (CGI-58), a protein with no lipolytic activity that increases ATGL activity to promote triacylglyerol hydrolysis (11,14,44). Perilipin and HSL upon hormonal stimu...

Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species

“…S7 shows the helical wheel representations (assuming that this part of the protein indeed folds into -helices) of Thus, the phospholipid composition is likely to vary spatially and temporally in the cell. Previous work by the Atshaves group seems to suggest that LDs coated with perilipin 3 contain a lipid monolayer with more unsaturated chains than the pool of LDs that are primarily coated with perilipin 1 (16). If this is indeed the case, then it is unlikely that the amphipathic -helix bundle of perilipin 3 is bound to the phospholipid monolayer.…”

“…Only limited data on the phospholipid composition of the LD monolayer is available, for a very limited set of cell types (16,17). Instead, we chose those lipids with various associate with the LD surface is crucial for our understanding of LD biology.…”

“…The phospholipid composition of the monolayer surrounding the LDs has not been clearly defined, but appears to consist mostly of phosphatidylcholine (PC) with a series of other phospholipids, diacylglycerol, and cholesterol (16,17).…”

Genetics of Lipid-Storage Management inCaenorhabditis elegansEmbryos

“…Acid sphingomyelinases are crucial in establishing a well-balanced level between SM and ceramide production in the plasma membrane (Goni and Alonso 2002;Jenkins et al 2009;Young et al 2013). However, SM has been discovered in lipid droplet fractions in a variety of different cell types (Hood and Patton 1973;Ishii et al 1995;Tauchi-Sato et al 2002;McIntosh et al 2010;Storey et al 2011). Moreover, SMs, PCs, and PIs are enriched in functionally active lipid droplets (Storey et al 2011).…”

“…However, SM has been discovered in lipid droplet fractions in a variety of different cell types (Hood and Patton 1973;Ishii et al 1995;Tauchi-Sato et al 2002;McIntosh et al 2010;Storey et al 2011). Moreover, SMs, PCs, and PIs are enriched in functionally active lipid droplets (Storey et al 2011). The conversion of SMs to ceramide causes structural changes in membranes, facilitating the formation of vesicles (Wheeler et al 2009;Mitsutake et al 2011).…”