Energy-dependent protein-triacylglycerol interaction in a cell-free system from 3T3-L1 adipocytes

Triacylglycerol is synthesized from the precursors sn-1,2-diacylglycerol and and palmitoyl-CoA in a reaction catalyzed by the microsomal enzyme diacylglycerol acyltransferase (EC 2.3.1.20). Isolated 3T3-L1 adipocyte microsomal vesicles from cells pulse-labeled with L-[35S]methionine were found to release microsomal proteins into a low density form during the synthesis of triacylglycerol. The proteins released, which represent a subset of those present in the labeled microsomes, include a 62-kDa protein found in high concentration in mature fat droplets. The formation of the triacylglycerol-protein complexes was dependent on time and temperature, was not stimulated by cytosol, and required ATP as well as diacylglycerol and palmitoyl-CoA. Only nucleoside triphosphates and not non-hydrolyzable analogues could replace ATP in the reaction. Unlike the enzyme reaction that measures the synthesis of triacylglycerol, the formation of low density membrane is thus dependent on ATP hydrolysis as well as enzyme substrates. The newly formed, low density particles are selectively enriched in triacylglycerol synthesized during the reaction as well as that synthesized prior to the reaction. The cell-free system described thus appears to represent an early adipogenic event leading to the lipid vacuoles found in mature adipocytes.