Figure 6

Fatty acid incorporation in ER-reduced lipid droplet subpopulations.

LDs and cytosol were isolated from CHO K2 cells and the LDs were further separated into three subpopulations using differential centrifugation (Materials and Methods). Fraction 3, the LD subpopulation nearly depleted of ER, was used for an in vitro fatty acid incorporation assay. The faction 3 LDs had first been incubated with [³H]OA and unlabeled OA at 4 °C for 6 h. (A) Requirement of ATP and cytosol for fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with 2 mM ATP alone or 100 μl cytosol in the presence or absence of 2 mM ATP at 37 °C for 2 h. (B) Requirement of ATP and CoA for fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with 2 mM CoA alone or the indicated concentrations of CoA (mM) in the presence or absence of 2 mM ATP at 37 °C for 2 h. (C–F) Time-dependent fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with the indicated concentration of ATP (μM) at 37 °C for 2 h in the presence of 100 μl cytosol (C), or with 2 mM ATP in the presence or absence of 2 mM CoA at 37 °C for the indicated times (D–F). After incubation, LDs were re-isolated, washed 3 times with Buffer B and lipids extracted with acetone:chloroform (2:1, v/v) and separated using TLC. The bands of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerol (TAG) were scraped from TLC plate and counted with scintillation fluid.