Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies

Abstract

We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call "lipid sails." TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.An entirely new view of the functions and composition of lipid bodies is currently emerging from studies on proteins associated with these organelles. Lipid bodies are clearly more than mere storage depots for superfluous neutral lipids in times of metabolic stress (1). Aside from their obvious involvement in lipid homeostasis, they appear also to partake exquisitely in cell signaling (2, 3), intracellular vesicle trafficking (3, 4), and disease (5, 6). Lipid bodies are metabolically active organelles in cells of virtually all animals, many plants, and even some prokaryotes (7,8).Several proteins with similar amino-terminal amino acid sequences and hence common structural motifs, the PAT (perilipin, adipophilin, tail-interacting protein of 47 kDa) family proteins, interact with lipid bodies in as yet unknown ways. PAT family proteins are believed to localize exclusively to the surface of lipid bodies and to be evolutionarily conserved (9 -12). Similarities in the structure of PAT family proteins and their targeting to and localization in lipid bodies are suggestive of a common underlying function in lipid homeostasis and/or lipid body biogenesis.Among the members of the PAT family proteins, the tailinteracting protein of 47 kDa (TIP47) is still of uncertain status. TIP47 seems to be involved in delivering mannose-6-phosphate receptors from endosomes to the trans-Golgi network (13,14) and, at the same time, to be a component of the surface of lipid bodies (10, 15). These two cellular compartments, lipid bodies and the late endosome sorting compartment, are currently thought to be completely divorced.Several controversial reports on the putative association of TIP47 with lipid droplets, the most common form of lipid body in -tagged TIP47 in Chinese hamster ovary cells transfected with a vector containing the coding sequence for murine TIP47. GFP-TIP47 appeared to be at the lipid droplet surface, despite the fact that TIP47 was also strongly expressed throughout the cytoplasm of the cells. Nevertheless, the authors conclud...

Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis

“…One study reported that adipophilin in transfected murine 1246 cells is preferentially associated with the plasma membrane and the nucleus (Gao and Serrero, 1999), although subsequent studies failed to confirm this finding (McManaman et al, 2003). Although past studies have relied largely on low resolution immunofluorescence microscopy and biochemical analysis of subcellular fractions, our recent findings from freeze-fracture immunocytochemistry indicate that PAT family proteins are not restricted to the lipid droplet surface as previously maintained, but also pervade the droplet core (Robenek et al, 2005a;Robenek et al, 2005b). Moreover, we demonstrated unequivocally with this approach that PAT family proteins are indeed integral components of the plasma membrane (Robenek et al, 2005c) where they appear to play a role in the accumulation of fatty acids into lipid droplets that are juxtaposed to the plasma membrane.…”

“…Adipophilin was immunolabeled using well-characterized mouse monoclonal antibody to a synthetic peptide representing the N-terminus (amino acids 5-27) of human adipophilin (AP125, Progen Biotechnik, Heidelberg, Germany) (Robenek et al, 2005b;Robenek et al, 2006). Irrelevant antibodies against Lamp-1, connexin43 and the inner nuclear membrane proteins LAP2␤ and emerin were used as controls.…”

Butyrophilin controls milk fat globule secretion

“…The monolayer is derived from the cytoplasmic leaflet of the ER, so its hydrophobic aspect revealed in concavely fractured globules is considered to be a P-face view. The complementary aspect seen in convexly fractured globules, which actually represents a view of the outermost aspect of the core, is referred to as the E-face equivalent (25,27).…”

“…Adipophilin and TIP47 are lipid droplet-associated proteins that are assumed to reside exclusively in the envelope of lipid droplets and to be involved in droplet formation, but both are also clearly residents of the lipid droplet core (25,26) and of the plasma membrane (27). Butyrophilin, a type I transmembrane glycoprotein with a cytoplasmic C-terminal tail, is concentrated in the apical plasma membranes of mammary epithelial cells (24, 28 -33).…”

Lipid homeostasis and the formation of macrophage-derived foam cells in atherosclerosis

“…TIP47 is located on the LD surface of Hela and other cell types, including human THP-1 macrophages (Robenek et al, 2005a(Robenek et al, , 2005b, but TIP47 expression is not stimulated by oxLDL. However, knockdown of TIP47 by siRNA specifically reduced cellular TG content but not cholesterol content, indicating that TIP47 is mainly involved in TG accumulation .…”