Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

Abstract

During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDphosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.

Fretting about fat: a new look at the lipid droplet surface and the roundabout role of Plin2 in cellular lipid storage. Focus on “Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis”

“…The likely physical association was also suggested in previous fluorescent ligand saturation binding assays by McIntosh and colleagues (9). Here, however, their use of FRET shows for the first time the direct binding of Plin2 with lipid droplet surface lipids.…”

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

“…Surprisingly, caveolins and flotillin, proteins commonly found in plasma membrane caveolae, were also detected (13,34,35,64,75,76,81). The presence of these proteins, along with cholesterol (9, 80) and sphingomyelin (48,53,57,68,101) in the LD phospholipid monolayer, gives rise to the hypothesis that localized areas may exist within the LD monolayer that on the basis of membrane structure attract proteins involved in lipolysis, much like the highly organized areas within the plasma membrane where lipid uptake/efflux occurs (18,30,62,79). The affinity of cholesterol for sphingolipids represents a driving force for selective recruitment and sequestering of proteins of similar function (18).…”

“…Furthermore, sterol efflux from LD revealed multiple dynamic cholesterol domains, one of which was similar to the small, rapid pools found in the plasma membrane. In addition, kinetic analysis of HDL-mediated 6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-hexanoyl} sphingosyl-phosphocholine (NBD-sphingomyelin) efflux from LD in living cells allowed resolution of multiple sphingomyelin pools, revealing a small, dynamic pool with a half-time of 11.8 min and another larger, relatively inert pool with a half-time on the order of hours to days (68). A comparison of LD and plasma membrane sphingomyelin dynamic pools revealed that the LD pool was smaller with faster efflux kinetics.…”

“…To extend the earlier discovery of dynamic sterol and sphingomyelin LD pools (9,68), the investigation herein examined how the membrane characteristics of the LD phospholipid monolayer affected LD protein targeting and function. The present work demonstrates for the first time the following novel insights.…”

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).…”