Identification of Two Regions in Apolipoprotein B100 that are Exposed on the Cytosolic Side of the Endoplasmic Reticulum Membrane

Abstract

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.

Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity

“…Neuro-2a cells transiently transfected with various apoE4 cDNA constructs were grown in serum-free MEM for 18-24 h, fixed in 3% paraformaldehyde, permeabilized for 45 min at room temperature with 500 units of Streptolysin-O (STP-O, Sigma) in BBII buffer (75 mM potassium acetate͞25 mM Hepes, pH 7.2) (for plasma membranes) or 0.5% Tween 20 in PBS (for plasma and intracellular organelle membranes) (51), and stained with polyclonal anti-apoE (1:4,000 dilution) or monoclonal anti-apoE (3H1, 1:200 dilution) and a fluorescein-coupled secondary antibody (Vector Laboratories) (25). Labeled cells were mounted in VECTASHIELD (Vector Laboratories) and viewed with a Radiance 2000 laser-scanning confocal system (Bio-Rad) that was mounted on an Optiphot-2 microscope (Nikon).…”

Nuclear Export of the Glucocorticoid Receptor Is Accelerated by Cell Fusion-dependent Release of Calreticulin

“…Analysis of Calreticulin and Calnexin Exposure-Selective permeabilization experiments were performed using a modification of the protocol of Du et al (31). Cells were plated onto 22-mm square coverslips, treated as described, and then fixed with 3% paraformaldehyde in PBS for 30 min at 4°C, followed by incubation with PBS containing 0.1 M glycine for 10 min at 20°C.…”

Cytosolic Components Are Required for Proteasomal Degradation of Newly Synthesized Apolipoprotein B in Permeabilized HepG2 Cells

“…Permeabilization of HepG2 Cells-The cell permeabilization procedures were described previously (23). In this study, we used digitonin to perforate the plasma membrane and saponin to perforate both plasma and intracellular membranes.…”

“…ApoB in cells was extracted with lysis buffer, immunoprecipitated with anti-human apoB antibody, and separated by SDS-PAGE. Proteins in the gel were transferred to polyvinylidene fluoride Immobilon-P membranes as described previously (23). ApoB and apoB-bound ubiquitin in the membrane were detected by anti-apoB and anti-ubiquitin antibodies and visualized with horseradish peroxidase-conjugated second antibodies and ECL luminol reagent.…”

“…Cell Culture-HepG2 cells were grown on collagen-coated tissue culture dishes (for labeling experiments) or coverslips (for immunocytochemistry) as described previously (23). Briefly, HepG2 cells were cultured in 24-or 6-well tissue culture plates with complete medium containing minimal essential medium with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum.…”