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J. Biol. Chem., Vol. 278, Issue 29, 27293-27300, July 18, 2003

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A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System^*

Denis Marchesan , Mikael Rutberg , Linda Andersson  , Lennart Asp , Thomas Larsson , Jan Borén , Bengt R. Johansson ¶ and Sven-Olof Olofsson  ||

From the Department of Medical Biochemistry and the Wallenberg Laboratory for Cardiovascular Research, the Swegene Proteomic Center, and the ^¶Department of Anatomy and Cell Biology, Göteborg University, SE-413 45 Göteborg, Sweden

We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TG-containing structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 x g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.

Received for publication, February 10, 2003 , and in revised form, April 29, 2003.

^* This work was supported by Grant 7142 from the Swedish Medical Research Council, the Swedish Heart and Lung Foundation, Novo Nordic Foundation, the Söderberg Foundation, Nestlé, and the Swedish Strategic Funds (National Network and Graduate School for Cardiovascular Research). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Wallenberg Laboratory, Sahlgrenska University Hospital/S, SE-413 45 Göteborg, Sweden. Tel.: 46-31-342 1956; Fax: 46-31-82 37 62; E-mail: Sven-Olof.Olofsson{at}medkem.gu.se.

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