Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

Abstract

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50 -100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. INTRODUCTIONLipid droplets (CLDs) consist of a neutral lipid core with a surrounding phospholipid monolayer (Murphy and Vance, 1999;Tauchi-Sato et al., 2002). CLDs are prominent in adipose cells and steroidogenic cells, but they also exist in other cell types. With the exception of a few cell types, CLDs have been considered as inert excess lipid deposits. However, recent studies have shown that a variety of proteins are localized in CLDs, suggesting that they may play more active functional roles than previously thought. In addition to PAT family proteins, enzymes involved in eicosanoid formation, enzymes for cholesterol synthesis, signaling proteins, caveolins, and Rab proteins have been reported in CLDs (Ozeki et al., 2005, and references therein). Proteomic studies identified many more proteins of both known and unknown functions in the CLD-rich fraction (Brasaemle et al., 2004;Fujimoto et al., 2004;Liu et al., 2004;Umlauf et al., 2004). The presence of functional proteins implies that CLDs are not mere lipid storage vessels but may be involved in various cellular activities.In hepatocytes, the triglycerides in CLDs are thought to be used for the synthesis of very low-density lipoprotein (VLDL) (Gibbons et al., 2000). Enzyme activities that hydrolyze and reesterify neutral lipids are found in CLDs and/or endoplasmic reticulum (ER), but the detailed mechanism by which the CLD content is mobilized and incorporated into lipoprotein particles is still unclear (Murphy, 2001). Apolipoprotein B (ApoB)-100, the primary protein component of VLDL, is assembled with lipids in the ER lumen, and further lipidation and maturation of the lipoprotein particles occur in the ER or the pre-Go...

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