Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B.
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.
Ohsaki
Yuki
Y
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. yohsaki@med.nagoya-u.ac.jp
Cheng
Jinglei
J
Fujita
Akikazu
A
Tokumoto
Toshinobu
T
Fujimoto
Toyoshi
T
D001055:Apolipoproteins B / Q000378:metabolism* / Q000648:ultrastructure; D001343:Autophagy; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008055:Lipids / Q000502:physiology*; D008079:Lipoproteins, VLDL / Q000096:biosynthesis; D008113:Liver Neoplasms; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
D001055:Apolipoproteins B; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008055:Lipids; D008079:Lipoproteins, VLDL; C063261:acetylleucyl-leucyl-norleucinal; D046988:Proteasome Endopeptidase Complex