Abstract
The mobilization of stored lipid by hormones is a fundamental function of fat cells, and there is strong evidence that perilipin (Plin), a lipid droplet scaffold, and adipose tissue triglyceride lipase (Atgl), a triglyceride-specific lipase, play critical roles. Previous work suggested that Abhd5, a protein activator of Atgl, coordinates with Plin in controlling basal and stimulated lipolysis; however, the underlying mechanism is controversial. The present experiments investigated protein trafficking and interactions among Plin, Atgl, and Abhd5 in live cells. The results demonstrate that Plin binds Abhd5 with high affinity and thereby suppresses the interaction of Abhd5 with Atgl. Sequestration of Abhd5 appears to a major mechanism by which Plin reduces basal lipolysis. Phosphorylation of Plin on serine 492 or serine 517 rapidly releases Abhd5 from Plin, allowing Abhd5 to directly interact with Atgl. Imaging experiments demonstrated that the Plin-dependent interaction of Abhd5 and Atgl occurs mainly, but not exclusively, on lipid droplets that contain Plin.
Highlights
Unlike hormone-sensitive lipase (HSL), adipose tissue triglyceride lipase (Atgl) is not a direct target of protein kinase A (PKA) phosphorylation, and the mechanism of its activation must be indirect
We identify the PKA phosphorylation sites on Plin that are necessary for releasing Abhd5 and further demonstrate that phosphorylation of these sites mediates the PKA-dependent interaction of Abhd5 with Atgl
To quantify the BiFC Atgl—Previous immunochemical analyses have shown that signals, the subcellular domain containing Plin-ECFP lipid droplets (LDs) was endogenous Plin and Abhd5 are colocalized on LDs in the basal defined for each frame using the autosegmentation tool of IPlabs, state, whereas Atgl is found both in the cytosol and on LDs (3, 9, and the net fluorescence intensities of 10)
Summary
Subcellular Colocalization of Fluorescently Tagged Proteins— Cos were transfected with Plin-EYFP, Abhd5-Cherry, and ECFP-Atgl, incubated with oleic acid (200 M complexed to bovine serum albumin) overnight to promote lipid droplet formation, and imaged live the day. Luciferase Protein Complementation Analysis—Cos or 3T3-L1 preadipocytes were grown in 24-well plates and transfected in quadruplicate with N- and C-luciferase fragments fused to Atgl and Abhd along with wild type- or phosphorylation-defective Plin or various controls as specified in the figure legends. 3T3-L1 cells were transfected with ECFP-Abhd and wild type or mutant Plin-EYFP, loaded with oleic acid overnight. In some experiments cells were transfected with complementary BiFC fragments fused to Atgl and E262K Abhd in the presence of wild type Plin-ECFP. FRET between ECFP-Abhd and Plin-EYFP was monitored in live 3T3-L1 cells before and after PKA activation with forskolin/IBMX.
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