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.
Highlights
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
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
The major finding of this study is that CGI-58, a protein that is highly expressed in adipocytes, binds to perilipin A on the surfaces of adipocyte lipid droplets
Summary
Materials—Fetal bovine serum, fatty acid-free bovine serum albumin, triolein, oleic acid, isobutylmethylxanthine (IBMX), insulin, dexamethasone, forskolin, and horseradish peroxidase-conjugated goat anti-rabbit IgG were purchased from Sigma. Stable Expression of CGI-58-GFP Chimeras in 3T3-L1 Cells—A murine CGI-58 cDNA was amplified out of the 3T3-L1 adipocyte library using oligonucleotide primers corresponding to the 5Ј-coding sequence lacking the first 6 amino acids (5Ј-GCTCTAGAGAGGAGGTGGACTCGGCAG-3Ј) and 3Ј-coding and non-coding sequences including the stop codon (5Ј-GCTCTAGAGGTTCTGTGTGCTCAGTCTAC-3Ј) with added XbaI restriction sites using the Expand High Fidelity PCR System (Roche Applied Science). Ecotropic retroviral stocks were produced in transfected 293T cells as described previously [26], and used to infect cultured 3T3-L1 preadipocytes; cells stably expressing the cDNA were selected for by resistance to geneticin at 0.6 mg/ml culture medium. Supernatants were incubated with polyclonal antibodies raised in rabbits against a recombinant N-terminal peptide of perilipin A [6] or control non-immune rabbit serum, and protein A-Sepharose at 4 °C overnight, washed extensively in IP solution, and eluted in Laemmli sample buffer prior to SDS-PAGE and immunoblotting
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