Identification of diverse lipid droplet targeting motifs in the PNPLA family of triglyceride lipases.

Members of the Patatin-like Phospholipase Domain containing Protein A (PNPLA) family play key roles in triglyceride hydrolysis, energy metabolism, and lipid droplet (LD) homoeostasis. Here we report the identification of two distinct LD targeting motifs (LTM) for PNPLA family members. Transient transfection of truncated versions of human adipose triglyceride lipase (ATGL, also known as PNPLA2), PNPLA3/adiponutrin, or PNPLA5 (GS2-like) fused to GFP revealed that the C-terminal third of these proteins contains sequences that are sufficient for targeting to LDs. Furthermore, fusing the C-termini of PNPLA3 or PNPLA5 confers LD localization to PNPLA4, which is otherwise cytoplasmic. Analyses of additional mutants in ATGL, PNPLA5, and Brummer Lipase, the Drosophila homolog of mammalian ATGL, identified two different types of LTMs. The first type, in PNPLA5 and Brummer lipase, is a set of loosely conserved basic residues, while the second type, in ATGL, is contained within a stretch of hydrophobic residues. These results show that even closely related members of the PNPLA family employ different molecular motifs to associate with LDs.

Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover.

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.

ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells.

Lipid droplets (LDs), also called adiposomes, are found in many eukaryotic cells, and are highly upregulated in lipid-storage cells, such as adipocytes. The mechanism by which adiposomes and their component neutral lipids are degraded is an important health issue with the rapidly spreading epidemic of obesity. Recently, a novel triglyceride lipase (adipose triglyceride lipase (ATGL)) that catalyses the initial step in triglyceride hydrolysis in adipocyte LDs was identified. Here, we show that ATGL also functions in non-adipocyte cells, and has an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a marked decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs, but is unable to decrease their size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. These results show that ATGL has an important role in LD/adiposome turnover in mammalian cells.