Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.