Studies on intracellular translocation of apolipoprotein B in a permeabilized HepG2 system.
Recent evidence suggests that the rate of apolipoprotein B-100 (apoB) translocation may be a key regulatory point in the production of apoB-containing lipoproteins. We have developed an in vitro system to measure the translocation rate of apoB in HepG2 cells. Intact cells were initially pretreated with oleate and N-acetyl-Leu-Leu-norleucinal to maximize the translocation rate while minimizing degradation. Cells were pulsed with [35S]methionine, chased (5-30 min), and then permeabilized with digitonin (75 microg/ml). Permeabilized cells were incubated with or without trypsin (200 microg/ml) for 10 min, and digestion was halted with soybean trypsin inhibitor (2 mg/ml). The rate of translocation was determined by comparing the amount of immunoprecipitable intact apoB in trypsin-treated cells with that in control cells at each time point. Under these conditions, two control proteins, alpha1-antitrypsin and transferrin, were fully protected from trypsin digestion, confirming the integrity of the secretory pathway in permeabilized cells. The percentage of apoB translocated steadily increased from 36% after 5 min to 71% after a 30-min chase (mean percentage, n = 3). A characteristic apoB fragmentation pattern resulted from trypsin digestion, and protected fragments of various size including N-terminal 60-70-kDa fragments were identified. Subcellular fractionation of the cells confirmed that the apoB pool protected from trypsin digestion was luminal in nature, confirming its translocation. ApoB translocation was significantly increased in oleate-treated cells compared with untreated cells. Inhibition of peptidylprolyl isomerase through the use of cyclosporin A and disruption of disulfide bond formation using dithiothreitol reduced the percentage of translocated apoB by 37 and 63%, respectively. Dithiothreitol induced specific changes in the pattern of protected apoB fragments, suggesting a conformational change in apoB that may hinder its translocation. Inhibition of N-linked glycosylation with tunicamycin did not significantly alter the rate of apoB translocation but appeared to stimulate its degradation. Together, the data suggest that the rate of apoB translocation across the membrane of the ER is determined by both lipid availability as well as the correct conformation of nascent apoB molecules.
Macri
J
J
Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
Adeli
K
K
D001055:Apolipoproteins B / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability; D016572:Cyclosporine / Q000494:pharmacology; D006801:Humans; D014357:Trypsin
D001055:Apolipoproteins B; D016572:Cyclosporine; D014357:Trypsin