Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in a nonhepatic cell line.
To explore the process of lipoprotein assembly, plasmids encoding truncated forms of apolipoprotein B (apoB) were transfected into Chinese hamster ovary (CHO) fibroblasts. (One, encoding apoB53, the N-terminal 53% of apoB100, can direct the assembly and secretion of lipoproteins when expressed in hepatoma cells, while the other, encoding the shorter apoB15, does not direct lipoprotein assembly.) Expression of apoB15 in CHO cells resulted in the accumulation of apoB15 protein in both medium and cells. In contrast, apoB was not detectable in medium or within CHO cells transfected with the plasmid encoding apoB53, despite the expression of apoB53 mRNA. ApoB53 did accumulate within transfected cells incubated with the thiol protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), suggesting that it is synthesized but completely degraded in the absence of the inhibitor. ApoB53 was not secreted despite its presence within ALLN-treated cells. Essentially all the apoB53 that accumulated in microsomes from ALLN-treated cells was associated with the membrane and was susceptible to degradation by exogenous trypsin, indicating exposure on the cytoplasmic face of the membrane. Thus, translocation of apoB53 across the endoplasmic reticulum membrane is blocked. However, the apoB53 bound to concanavalin A, suggesting that it is glycosylated and therefore partly exposed to the lumen as well. ApoB requires a unique process, not expressed in CHO fibroblasts, for its complete translocation and entrance into the secretory pathway. This process might account for the inability of abetalipoproteinemic patients to secrete apoB.
Thrift
R N
RN
Atherosclerosis Research Center, University of Colorado Health Sciences Center, Denver 80262.
Drisko
J
J
Dueland
S
S
Trawick
J D
JD
Davis
R A
RA
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D003208:Concanavalin A / Q000378:metabolism; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D001055:Apolipoproteins B; D012333:RNA, Messenger; D003208:Concanavalin A