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. 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. Apolipoprotein A-V (apoA-V), first described in 2001 (1Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox C.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (800) Google Scholar, 2Van der Vliet H.N. Sammels M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), has emerged as an important modulator of triglyceride (TG) metabolism (3Wong K. Ryan R.O. Characterization of apolipoprotein A-V structure and mode of plasma triacylglycerol regulation.Curr. Opin. Lipidol. 2007; 18: 319-324Crossref PubMed Scopus (37) Google Scholar). The protein is synthesized solely by the liver and is found in plasma associated with HDL and VLDL. Newly synthesized apoA-V consists of 366 amino acids, where the first 23 residues form the cleavable signal peptide. The mature, secreted form of apoA-V consists of 343 residues (39 kDa). The protein is extremely hydrophobic and not readily soluble in aqueous medium; however, once bound to lipid, it is soluble and stable (4Beckstead J.A. Oda M.N. Martin D.D.O. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9416-9423Crossref PubMed Scopus (71) Google Scholar). We recently reported that apoA-V, overexpressed in hepatoma cell lines, associates with cytoplasmic lipid droplets despite being synthesized with a signal peptide (5Shu X. Chan J. Ryan R.O. Forte T.M. ApoA-V association with intracellular lipid droplets.J. Lipid Res. 2007; 48: 1445-1450Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Although the protein was poorly secreted from hepatoma cells, it was found in association with lipoproteins in conditioned medium. In hepatocytes, lipid droplets are a storage form of neutral lipids (particularly TG) that may be used for production of VLDL. Since it is conceivable that apoA-V lipid droplet association may be relevant to plasma TG homeostasis, we explored these observations further to determine structural elements within apoA-V that are involved in lipid droplet association and apoA-V secretion. It was previously proposed that the C-terminal segment of apoA-V is required for lipid binding (6Weinberg R.B. Cook V.R. Beckstead J.A. Martin D.D.O. Gallagher J.W. Shelness G.S. Ryan R.O. Structure and interfacial properties of human apolipoprotein A-V.J. Biol. Chem. 2003; 278: 34438-34444Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and this proposal is supported by the in vitro observation that the apoA-V(1-292) variant had lower lipid binding ability than the full-length protein (7Beckstead J.A. Wong K. Gupta V. Wan C-P.L. Cook V.R. Weinberg R.B. Weers P.M.M. Ryan R.O. The effect of C-terminal truncation on the structural and lipid binding properties of apolipoprotein A-V.J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In human subjects with a mutant form of apoA-V, Q139X, in which putative hydrophobic, amphipathic helices of the C terminus are deleted, association with lipoproteins in plasma did not occur (8Marçais C. Verges B. Charriere S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment.J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (140) Google Scholar). These observations strongly suggest a role for the C terminus in lipid binding. In the present study, several C-terminal apoA-V truncation variants were expressed in hepatoma cells to evaluate how much of the C-terminal region is required for homing to lipid droplets and/or lipoprotein association. In keeping with the results obtained in human subjects carrying the Q139X apoA-V variant, apoA-V(1-146) described in this study did not associate with lipoproteins in conditioned medium. The latter variant was also unable to associate with lipid droplets, thus suggesting that the hydrophobic C-terminal region of this protein is required for lipid binding. Oleic acid (OA) and albumin were from Sigma. DMEM, horse serum, G418, and trypsin-EDTA were purchased from Invitrogen. FBS was from HyClone. Nile Red and 4′,6-diamino-phenylindole (DAPI) were from Molecular Probes. Polyclonal rabbit anti-human apoA-V antibody was a kind gift from Dr. Robert J. Konrad (Eli Lilly and Co.). Monoclonal mouse anti-adipocyte differentiation-related protein (ADRP) antibody was from Progen Biotechnik. Rat hepatoma McA-RH7777-A18 (McA-RH) cells stably transfected with human apoB-100 (kindly provided by Dr. Zemin Yao, University of Ottawa) were cultured in DMEM containing 10% FBS, 10% horse serum, and 200 μg/ml G418. Cells were passaged every 4 days. Cells were transfected using Lipofectamine 2000 (Invitrogen). DNA amplification was carried out using a plasmid template harboring the entire apoA-V coding region (a gift from Dr. Len Pennacchio, Lawrence Berkeley National Laboratory). Primers were designed to amplify full-length apoA-V and indicated truncations (Fig. 1). The amplification products were cloned into pcDNA3.1(+) (Invitrogen) or pEGFP-N1 (Clontech) in the case of expression of green fluorescent protein (GFP) fusions. The GFP tag was appended to the C terminus of the protein. For fluorescence microscopy, McA-RH cells were grown on poly-l-lysine coverslips (BD Biosciences). After transfection with apoA-V or apoA-V-GFP constructs, cells were transferred to growth medium supplemented with 0.8 mM OA for 6 h. For immunocytochemistry, cells were fixed with 4% paraformaldehyde in PBS and processed as described (9Tran K. Thorne-Tjomsland G. DeLong C.J. Cui Z. Shan J. Burton L. Jamieson J.C. Yao Z. Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells.J. Biol. Chem. 2002; 277: 31187-31200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Cells were washed with PBS, and lipid droplets were stained with Nile Red as described (10Greenspan P. Mayer E.P. Fowler S.D. Nile red: a selective fluorescent stain for intracellular lipid droplets.J. Cell Biol. 1985; 100: 965-973Crossref PubMed Scopus (1856) Google Scholar); nuclei were stained with DAPI. To visualize ADRP, fixed cells were permeabilized and subsequently incubated with mouse anti-ADRP (1:10 dilution) followed by incubation with goat anti-mouse Alexa 594 secondary antibody. Images were captured with a LSM 510 Meta UV/Vis confocal microscope. McA-RH cells were grown on poly-l-lysine-coated six-well plates (BD Biosciences). Twenty-four hours after transfection, medium was replaced with DMEM plus 10% FBS and 0.8 mM OA. After 16 h, cells were washed with PBS and incubated with serum-free medium for 24 h. Subsequently, conditioned medium was collected, cell monolayer was rinsed with PBS, and the cells were lysed with Cell Lysis Buffer (Cell Signaling Technology). To determine whether full-length apoA-V or apoA-V(1-146) associated with lipoproteins upon secretion into the cell medium, conditioned medium was adjusted to density 1.21 g/ml by the addition of solid NaBr. The sample was then centrifuged at 100,000 g for 5.5 h in a Beckman TL100 centrifuge. The lipoprotein-rich d ⩽ 1.21 g/ml fraction and the d > 1.21 g/ml lipoprotein-poor fraction were harvested by aspiration as described by Lindgren, Jensen, and Hatch (11Lindgren F.T. Jensen L.C. Hatch F.T. The isolation and quantitative analysis of serum lipoproteins.In Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. G. J. Nelson, editor. Wiley-Interscience, New York. 1972; : 181-274Google Scholar). We previously reported that, in OA-supplemented cultured hepatoma cells, ectopically expressed full-length apoA-V homes to lipid droplets coating their surfaces (5Shu X. Chan J. Ryan R.O. Forte T.M. ApoA-V association with intracellular lipid droplets.J. Lipid Res. 2007; 48: 1445-1450Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Since OA supplementation drives intracellular lipid accumulation and new lipid droplet formation, we sought to determine whether OA supplementation is required for the lipid droplet association of transfected apoA-V. To examine this question, an apoA-V-GFP fusion protein was expressed in McA-RH cells cultured with or without OA supplementation. Lipid droplet association was then examined by confocal microscopy. As seen in Fig. 2, in the absence of OA, lipid droplets were fewer in number and smaller in size. Despite this, however, transfected apoA-V-GFP localized to lipid droplets in a manner similar to that seen in cells supplemented with OA. Recently, Beckstead et al. (7Beckstead J.A. Wong K. Gupta V. Wan C-P.L. Cook V.R. Weinberg R.B. Weers P.M.M. Ryan R.O. The effect of C-terminal truncation on the structural and lipid binding properties of apolipoprotein A-V.J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) reported that the apoA-V(1-292) truncation variant (at the site of a unique tetra-proline sequence) can bind dimyristoylphosphatidylcholine vesicles but with lower binding efficiency than full-length apoA-V. These authors also showed that a 51 amino acid peptide encompassing residues 293–343 (the deleted portion of the protein) was highly efficient in binding to dimyristoylphosphatidylcholine. Taken together, these results suggested that this region of apoA-V is important in lipid binding. To test whether deletion of these residues, as well as other more extensive C-terminal truncations, alters apoA-V association with lipid droplets, plasmid vectors encoding truncated apoA-V-GFP variants were generated (Fig. 1). The latter approach provides new insights into how much of the C-terminal domain is required for lipid droplet and lipoprotein association. Following transfection into McA-RH cells, the extent of association with lipid droplets was determined by confocal microscopy (Fig. 3A). Consistent with our earlier report (5Shu X. Chan J. Ryan R.O. Forte T.M. ApoA-V association with intracellular lipid droplets.J. Lipid Res. 2007; 48: 1445-1450Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), full-length apoA-V associates with lipid droplets in the cell (Fig. 3A, top of apoA-V to lipid droplets is not the of the GFP fusion we previously showed (5Shu X. Chan J. Ryan R.O. Forte T.M. ApoA-V association with intracellular lipid droplets.J. Lipid Res. 2007; 48: 1445-1450Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) that without this fluorescent tag the protein associated with lipid droplets. deletion of 51 amino from the C terminus of apoA-V to lipid droplet association apoA-V-GFP truncation variants were transfected into McA-RH cells, and examined for their ability to associate with lipid droplets. The variant (Fig. and the apoA-V(1-191) variant not associated with intracellular lipid droplets, while apoA-V(1-146) did not. In the latter case (Fig. a fluorescent protein was and lipid droplets were smaller in size and the The of apoA-V in association with lipid droplets the of whether the N-terminal signal peptide is required to the protein to lipid droplets. To examine whether this 23 amino acid N-terminal M.G. Konrad N-terminal protein and further of the novel apolipoprotein in human Chem. PubMed Scopus Google Scholar) a role in apoA-V a plasmid encoding apoA-V-GFP that a signal was transfected into McA-RH cells and the intracellular of the protein was by confocal microscopy (Fig. 3A, compared with cells transfected with full-length apoA-V, present in the of fluorescence were ADRP, also as is the lipid protein in J. M. J. M. S. T. of in the lipid fraction from human cell PubMed Scopus Google Scholar). we whether apoA-V and on lipid droplets and whether the variant can this association. that full-length apoA-V and on the of lipid droplets in McA-RH studies with cells incubated in the absence of apoA-V (Fig. suggest that of apoA-V not trafficking to lipid droplets. of the variant results in with (Fig. lipid droplets are with Fig. 3A, suggesting that of the signal peptide did not to intracellular In cultured hepatoma cells, it is that apoA-V is not as efficiently secreted as other as or (5Shu X. Chan J. Ryan R.O. Forte T.M. ApoA-V association with intracellular lipid droplets.J. Lipid Res. 2007; 48: 1445-1450Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The and lipid binding ability of apoA-V may to this The that transfected apoA-V(1-146) not associate with lipid droplets that this variant is hydrophobic and has a lower lipid on this, it was that apoA-V(1-146) be more efficiently secreted from transfected hepatoma cells compared with full-length apoA-V or To test this, full-length apoA-V, apoA-V(1-292), and apoA-V(1-146) GFP fusion protein were transfected into McA-RH After 24 h of in serum-free medium, conditioned medium was and cell lysates were of cell and conditioned medium were then to to determine the of apoA-V (Fig. of full-length apoA-V-GFP and were in the cell a much of was in the medium. without the GFP tag showed a similar not the C-terminal truncations, of was in the cell (Fig. In this a GFP tag was not full-length apoA-V a GFP tag was for and that the of protein in cell versus medium was similar to that seen with Since transfected apoA-V(1-146) not associate with intracellular lipid droplets, we sought to determine whether the secreted protein associates with lipoproteins in conditioned medium. medium was and the d ⩽ 1.21 g/ml and d > 1.21 g/ml were full-length apoA-V was in association with lipoproteins, apoA-V(1-146) was in the lipoprotein-poor fraction (Fig. The observation that a portion of the full-length apoA-V expressed in transfected hepatoma cells was in the lipoprotein-poor fraction of conditioned medium observations that apoA-V was found associated with lipoproteins in human J.H. M. A. Konrad The novel apolipoprotein is present in human serum, is associated with and and at very low compared with other Chem. 2005; PubMed Scopus Google Scholar) and mouse L. X. Konrad Ryan R.O. Forte T.M. of apolipoprotein A-V on plasma lipoprotein size and in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) plasma and that apoA-V is not soluble at in a form (4Beckstead J.A. Oda M.N. Martin D.D.O. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9416-9423Crossref PubMed Scopus (71) Google Scholar). In the present it is conceivable that apoA-V in the d > 1.21 g/ml fraction may a of It is also that, to the lipoprotein secretion efficiency of these cells with of apoA-V, is lipid for of the apoA-V to bind to To whether the apoA-V in the d > 1.21 g/ml fraction is of binding lipoproteins, the lipoprotein-deficient fraction from conditioned medium was incubated with the HDL fraction obtained from density showed that apoA-V transferred to the HDL When the was with conditioned medium containing binding to HDL was not not Lipid droplets are of neutral lipids or and as a lipid storage C. C.J. ADRP, and other that associate with intracellular neutral lipid droplets in Biol. PubMed Scopus Google Scholar, The of structural lipid droplet of lipid droplets and of Lipid Res. 2007; 48: Full Text Full Text PDF PubMed Scopus Google Scholar). The of lipid droplets is of a monolayer with several associated lipid droplets are in can be found in cell on the lipid ADRP, and to as has revealed structural with the of amphipathic C. J.H. Structure of a lipid droplet the Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of provided for a and of C-terminal domain revealed a similar to the N-terminal of although lipid droplets are of in cells but to in than lipoproteins, both a a hydrophobic and a monolayer of on this, it is to that the binding of with lipid droplets may be to that and lipoprotein The of and are to upon lipid hydrophobic lipid binding in the Apolipoprotein Chem. PubMed Google Scholar, structure of human apolipoprotein insights into effect PubMed Scopus Google Scholar). the of the N-terminal domain and the C-terminal a similar may association with the of lipid droplets. not a as lipid and and An may be a recently apolipoprotein of which is reported to associate with lipid droplets in M. R. P. A. J. M. X. P. et a novel is an by in human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on this, it is not that apoA-V can associate with lipid droplets. The observation that apoA-V associates with lipid droplets is to expression as a containing a 23 amino acid signal peptide. this N-terminal the synthesized protein to the for the from the not M. R. P. A. J. M. X. P. et a novel is an by in human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), is how to a this In this of the of lipid droplet may a Lipid droplets form in to in of the with neutral lipid of the lipid droplets are to from the an the of this to the it is conceivable that lipid within this may associate with lipid droplets and with as from the the it is that in lipid droplet S. M. K. T. C. A. and of associated with lipid droplet protein a novel protein that on lipid PubMed Scopus Google Scholar) as well as of apoA-V der Vliet H.N. Sammels M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). apoA-V lipid droplet or In the present study, we that the lipid droplet association of apoA-V is not upon OA supplementation of but the C-terminal portion of the protein. full-length apoA-V but also C-terminal truncation apoA-V(1-292), apoA-V(1-237), and their ability to associate with lipid droplets transfection into hepatoma of lipid droplet homing ability the C terminus was truncated to of 343 amino in full-length the of lipid droplet full-length apoA-V and the apoA-V(1-292) variant that, in the latter despite lipid binding (7Beckstead J.A. Wong K. Gupta V. Wan C-P.L. Cook V.R. Weinberg R.B. Weers P.M.M. Ryan R.O. The effect of C-terminal truncation on the structural and lipid binding properties of apolipoprotein A-V.J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), amphipathic elements to effect binding of the protein to lipid droplets. the other the apoA-V(1-146) variant, in which a putative highly hydrophobic region has deleted, did not associate with intracellular lipid droplets. Although apoA-V(1-146) was efficiently secreted by the cells, it did not associate with lipoproteins, with inability to associate with lipid droplets. The of this variant is similar to that of a Q139X apoA-V mutant found in humans (8Marçais C. Verges B. Charriere S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment.J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (140) Google Scholar), in which the latter protein was found solely in the lipoprotein-deficient fraction of results obtained from studies of apoA-V(1-146) that the C-terminal region of apoA-V is for lipoprotein association. the transfection studies that the absence of the C-terminal not the observation of truncated apoA-V in subjects Q139X apoA-V. The in lipid droplet cells transfected with apoA-V with or without signal peptide suggest that this N-terminal may be for lipid droplet association. The that the signal peptide on apoA-V is can be on the of data with which a signal peptide to that present on full-length apoA-V. that the signal peptide to this protein to a The that the of a portion of the full-length apoA-V in these cells are not but may be to the of the protein. In the for this it is conceivable that the signal of synthesized apoA-V to the may in of the protein to the site of lipid droplet that lipid droplet assembly in the into the and to a apoA-V the with the site of droplet In the absence of a signal apoA-V to lipid droplet where the latter Although to lipid binding may the of the lipid droplets. The however, not to be to cell with ADRP, a lipid protein in hepatocytes, is In several are to be associated with reduced plasma apoA-V and plasma TG L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. apolipoprotein human plasma triglyceride 2002; PubMed Google Scholar). which is found in the signal peptide domain of the was to with the of apoA-V protein J. W. L. of the of Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the latter study, of by reduced the of fusion protein secreted into cell medium. In the present study, deletion of the signal peptide resulted in an inability of apoA-V to be suggesting that the signal peptide is required to extracellular trafficking of the protein. In the data for an intracellular mode of of apoA-V. These are in keeping with the very low plasma of this secretion and the observation that apoA-V is in to the observation that the C-terminal is for lipid droplet association, as well as lipoprotein association, that this region of the protein may be essential for the of TG by this protein. in studies with truncated apoA-V variants this to be The authors Dr. for HDL from

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