Abstract
Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.
Highlights
The very low density lipoprotein (VLDL)1 synthesized in the liver carries various amounts of triacylglycerol (TG) in the neutral lipid core surrounded by phospholipids, cholesterol, and apolipoproteins
ApoB100 polypeptide remains associated with the endoplasmic reticulum (ER) membranes [11, 12], and the resulting lipoprotein particle has buoyant density resembling that of high density lipoproteins (HDL) [13, 14]
We have determined the path through which the membrane-bound nascent apoB100 polypeptides are converted into buoyant VLDL
Summary
Materials—Glycerol [14C]trioleate (57 mCi/mmol), [35S]methionine/ cysteine (1000 Ci/mmol), [3H]palmitic acid (52 Ci/mmol), protein ASepharoseTM CL-4B beads, and horseradish peroxidase-linked antimouse or anti-rabbit IgG antibodies were purchased from Amersham Biosciences. Pulse-Chase Experiments—In pulse-chase experiments where lumenal apoB100 particles of different subcellular fractions were determined, the cells in two 100-mm dishes were labeled with [35S]methionine/cysteine (200 Ci/ml in 3 ml of methionine- and cysteine-free DMEM containing 20% FBS and 0.4 mM oleate) for 20 min. In experiments where transit of newly synthesized apoB100 along the secretory pathway was determined, the cells were pulse-labeled for 10 min, washed, and incubated with chase medium containing 10 M cycloheximide for 10, 20, 40, 80, and 120 min. In experiments where intracellular distribution of membrane- and lumen-associated apoB100 was determined, the cells were pulse-labeled for 20 min and incubated with chase medium for 0, 15, 30, and 45 min. Transmission Electron Microscopy—The cells were cultured in normal culture medium on MILLICELLTM-CM insert membranes precoated with fibronectin for 20 h and incubated for additional 2 h with fresh DMEM containing 20% FBS and 0.4 mM oleate. The protein was determined using the BCA protein assay kit (Pierce)
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