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© The Rockefeller University Press, 0021-9525/1998//585 $5.00
The Journal of Cell Biology, Volume 141, Number 3, , 1998 585-599

Articles

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



Xiaobo Du*, J. Daniel Stoops*, James R. Mertz§, C. Michael Stanley{ddagger}, and Joseph L. Dixon*

* Department of Food Science and Human Nutrition and {ddagger} Department of Biological Sciences, University of Missouri, Columbia, Missouri 65211; and § CUNY Medical School, New York, New York 10031

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.

Abbreviations used in this paper: ALLN, acetyl-leucyl-leucyl-norleucinal; ApoB, apolipoprotein B-100; ERGIC, ER-to-Golgi intermediate compartment; LAMP-1, lysosomal-associated membrane protein-1; LDL, low-density lipoprotein; MAP, multiple antigenic peptide; OA, oleate; PK, proteinase K; STP-O, streptolysin-O; TIPI, total integrated pixel intensity.

Preliminary versions of this work were presented at the Experimental Biology 95 meeting (Atlanta, GA, April 1995, abstract 4460) and the American Heart Association meeting (Anaheim, CA, November 1995, abstract 0787).

Address all correspondence to Joseph L. Dixon, Department of Food Science & Human Nutrition, University of Missouri-Columbia, 122 Eckles Hall, Columbia, MO 65211. Tel.: 573-882-4113; Fax: 573-882-0596; E-mail: jdixon{at}showme.missouri.edu


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