Intracellular lipid droplet targeting by apolipoprotein A-V requires the C-terminal and signal peptide segments

Xiao Shu, Robert O. Ryan, and Trudy M. Forte

Children's Hospital Oakland Research Institute, Oakland, CA 94609

Corresponding Author: tforte{at}chori.org

Expression of apolipoprotein (apo) A-V in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent fusion protein were cultured in media that was not supplemented with oleic acid, intracellular lipid droplet size and number were reduced compared to that of cells supplemented with oleic acid. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated apoA-V variants that associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium although it associated with lipid droplets. Taken together, the data suggest that the C-terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.

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