Identification of two regions in apolipoprotein B100 that are exposed on the cytosolic side of the endoplasmic reticulum membrane.
Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)- and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O-treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O-treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221-3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O-permeabilized cells. Staining with this antibody was similar in STP-O- and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690-797 (79-91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878-2148 (D7.2) and 3214-3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O-treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O-treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.
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X
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Department of Food Science and Human Nutrition, University of Missouri, Columbia, Missouri 65211, USA.
Stoops
J D
JD
Mertz
J R
JR
Stanley
C M
CM
Dixon
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin; D019286:Endopeptidase K / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D007553:Isotope Labeling; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes; D008562:Membrane Glycoproteins / Q000032:analysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011817:Rabbits; D012756:Sheep; D013194:Staining and Labeling; D014316:Tritium; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D014316:Tritium; D019286:Endopeptidase K; D004072:Digitonin