title: The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells.
abstract: The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.
authors.affiliation: Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
authors.forename: Junji
authors.initials: J
authors.lastname: Yamaguchi
authors.forename: Mary V
authors.initials: MV
authors.lastname: Gamble
authors.forename: Donna
authors.initials: D
authors.lastname: Conlon
authors.forename: Jun-shan
authors.initials: JS
authors.lastname: Liang
authors.forename: Henry N
authors.initials: HN
authors.lastname: Ginsberg
mesh_terms: D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D019301:Oleic Acid / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D020126:Brefeldin A; D019301:Oleic Acid; D015739:Nocodazole