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Journal of Lipid Research, Vol. 49, 1670-1676, August 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment^*

Xiao Shu^*,, Robert O. Ryan^*, and Trudy M. Forte^1,*

^* Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, CA 94609
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720-3104

^* This study was supported by Grant HL-073061 from the National Institutes of Health.

Published, JLR Papers in Press, April 30, 2008.

¹ To whom correspondence should be addressed. e-mail: tforte{at}chori.org

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.

Supplementary key words McA-RH7777 cells • confocal fluorescence microscopy • apolipoprotein A-V carboxyl-terminal truncation variants • lipoprotein association • apolipoprotein A-V-deficient high density lipoprotein • adipocyte differentiation-related protein • signal peptide

Abbreviations: ADRP, adipocyte differentiation-related protein; apoA-V, apolipoprotein A-V; DAPI, 4',6-diamino-phenylindole; GFP, green fluorescent protein; McA-RH, McA-RH7777; OA, oleic acid; TG, triglyceride

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				T. M. Forte, X. Shu, and R. O. Ryan The ins (cell) and outs (plasma) of apolipoprotein A-V J. Lipid Res., April 1, 2009; 50(Supplement): S150 - S155. [Abstract] [Full Text] [PDF]