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. 2004 Oct 1;279(40):42062-71.
doi: 10.1074/jbc.M407462200. Epub 2004 Aug 2.

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes

Vidya Subramanian  1 , Alexis Rothenberg, Carlos Gomez, Alex W Cohen, Anne Garcia, Sucharita Bhattacharyya, Lawrence Shapiro, Georgia Dolios, Rong Wang, Michael P Lisanti, Dawn L Brasaemle
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Free article

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes

Vidya Subramanian et al. J Biol Chem. .
Free article

Abstract

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.

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