Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets 
with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This 
study addresses the following two questions. Where do lipid droplets emerge, and 
how are their coat proteins recruited? We show that perilipin 3- and perilipin 
4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking 
membrane trafficking with AlF(4)(-) during fatty acid-induced triacylglycerol 
synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF(4)(-) 
activates adenylate cyclase, did not redistribute perilipin 3, but when added 
together with AlF(4)(-) perilipin 3 was recruited to lipid droplets rather than 
the ER. Thus inhibiting trafficking with AlF(4)(-) redistributed perilipin 3 
differently under conditions of triacylglycerol synthesis (fatty acid addition) 
versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated 
mechanism. We tested whether diacylglycerol (DG), the immediate precursor of 
triacylglycerol and its first hydrolytic product, affects the distribution of 
perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the 
perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. 
Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. 
Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with 
triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER 
enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated 
perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove 
perilipin 4 and 5 onto the ER. Together the data suggest that these lipid 
droplet proteins are recruited to DG-enriched membranes thereby linking lipid 
coat proteins to the metabolic state of the cell.