Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis.

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 ...
cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.
Mesh Terms:
Adipocytes, Bacterial Proteins, Carrier Proteins, Cell Line, Fatty Acid-Binding Proteins, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Humans, In Vitro Techniques, Lipid Metabolism, Luminescent Proteins, Multiprotein Complexes, Mutagenesis, Site-Directed, Protein Binding, Recombinant Fusion Proteins, Sterol Esterase, Transfection
J. Biol. Chem.
Date: Dec. 10, 2004
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