Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment.
The expression of apolipoprotein A-V (apoA-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 protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. 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 that apoA-V variants 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, these 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.
Shu
Xiao
X
Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
Ryan
Robert O
RO
Forte
Trudy M
TM
D000595:Amino Acid Sequence; D000818:Animals; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins / Q000235:genetics / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008074:Lipoproteins / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D014407:Tumor Cells, Cultured
C514818:Apoa5 protein, rat; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins; D008055:Lipids; D008074:Lipoproteins; D021382:Protein Sorting Signals