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Vol. 17, Issue 6, 2674-2683, June 2006

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

Yuki Ohsaki^*, Jinglei Cheng^*, Akikazu Fujita^*, Toshinobu Tokumoto, and Toyoshi Fujimoto^*

*Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; and Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan

Submitted July 21, 2005; Revised March 22, 2006; Accepted March 24, 2006
Monitoring Editor: Jeffrey Brodsky

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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Yuki Ohsaki ( yohsaki{at}med.nagoya-u.ac.jp) or Toyoshi Fujimoto ( tfuijmot{at}med.nagoya-u.ac.jp)

Abbreviations used: ADRP, adipose differentiation-related protein; ALLN, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; ApoB, apolipoprotein B; CLD, cytoplasmic lipid droplets; 3-MA, 3-methyladenine; VLDL, very low-density lipoprotein.

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