A phospholipase D-dependent process forms lipid droplets containing caveolin, adipocyte differentiation-related protein, and vimentin in a cell-free system.
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.
Department of Medical Biochemistry and the Wallenberg Laboratory for Cardiovascular Research, Göteborg University, SE-413 45 Göteborg, Sweden.
Denis
D
Marchesan
Mikael
M
Rutberg
Linda
L
Andersson
Lennart
L
Asp
Thomas
T
Larsson
Jan
J
Borén
Bengt R
BR
Johansson
Sven-Olof
SO
Olofsson
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins / Q000378:metabolism*; D002474:Cell-Free System; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism; D000070780:Perilipin-2; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D014280:Triglycerides / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism*
C495541:Cav1 protein, mouse; C495537:Cav1 protein, rat; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins; D008565:Membrane Proteins; D000070780:Perilipin-2; D014280:Triglycerides; D014746:Vimentin; D010739:Phospholipase D