Distribution, transport, and degradation of apolipoprotein B-100 in HepG2 cells.
The transport of apolipoprotein B (apoB) between the endoplasmic reticulum (ER) and Golgi was studied in puromycin-synchronized HepG2 cells, using an antibody that could distinguish between apoB in ER and Golgi compartments. In cells with normal ER-to-Golgi transport, both albumin and apoB colocalized throughout the ER and appeared as intense, compact signals in Golgi. When ER-to-Golgi transport was blocked with brefeldin A, apoB and albumin remained colocalized in the ER network and three-dimensional constructed images showed more intense signals for both proteins in a central, perinuclear region of the ER. When protein synthesis was stopped in cells with brefeldin A-inhibited ER-to-Golgi transport, apoB degradation was visualized as a homogeneous decrease in fluorescence signal intensity throughout the ER that could be slowed with clasto-lactacystin beta-lactone, a proteasome inhibitor. Incubation of cells with CP-10447, an inhibitor of microsomal triglyceride transfer protein, inhibited apoB, but not albumin, transport from ER to Golgi. Nanogold immunoelectron microscopy of digitonin-permeabilized cells showed proteasomes in close proximity to the cytosolic side of the ER membrane. Thus, newly synthesized apoB is localized throughout the entire ER and degraded homogeneously, most likely by neighboring proteasomes located on the cytosolic side of the ER membrane. Although albumin is colocalized with apoB in the ER, as expected, it was not targeted for ER-associated proteasomal degradation.
Sakata
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Dalton Cardiovascular Research Center, University of Missouri, Research Park, Columbia, MO 65211.
Phillips
T E
TE
Dixon
J L
JL
D000418:Albumins / Q000378:metabolism; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D002463:Cell Membrane Permeability; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004072:Digitonin / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000418:Albumins; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D009097:Multienzyme Complexes; D020126:Brefeldin A; D011691:Puromycin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004072:Digitonin