IDENTIFICATION OF 2 REGIONS IN APOLIPOPROTEIN-B-100 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 p athway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing spe cific domains of apoB in streptolysin-O (STP-O)- and saponin-permeabil ized HepG2 cells. Lumenal epitopes on marker proteins in secretory pat hway compartments (p63, p53, and galactosyltransferase) were not stain ed by antibodies in STP-O-treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not per forated in STP-O-treated cells. An anti-apoB peptide antibody (B4) rec ognizing amino acids 3221-3240 caused intense staining in close proxim ity to the nuclear membrane, and less intensely throughout the secreto ry pathway in STP-O-permeabilized cells. Staining with this antibody w as similar in STP-O- and saponin-treated cells, indicating that this e pitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar resul ts indicating a cytosolic orientation were obtained with monoclonal an tibody CC3.4, which recognizes amino acids 690-797 (79-91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibo dies 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-treat ed cells. The anti-LDL and B1B6 antibodies produced almost identical p unctate patterns in STP-O-treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1 B6 epitope of apoB is exposed on the surface of the lysosome. The resu lts identify two specific regions in apoB that are exposed to the cyto sol in the secretory pathway.