Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Abstract

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatmentinduced 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. Pulsechase 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.

Presecretory oxidation, aggregation, and autophagic destruction of apoprotein-B: A pathway for late-stage quality control

“…full lipidation of apoB-lipoproteins to VLDL buoyancy occurs post-ER (35)(36)(37). Also, given the conspicuous physical barriers against translocating large, extensively lipidated apoBlipoproteins out of the secretory pathway, which would be required for the proteasome to have access to them, the autophagosomal/lysosomal system may be the only plausible mechanism for late-stage quality control of these particles.…”

“…Steady-state labeling of hepatic proteins. After the pretreatments described in the figure legends, endogenous proteins were metabolically labeled to steady-state (49) by incubating hepatocytes at 37°C for 3 h in met/cys-free DMEM-complete (met/cys-free high-glucose DMEM with 0.5% FBS, 0.5% horse serum, 1% streptomycin/penicillin, and 1% L-glutamine) supplemented with 100 Ci [ 35 After the labeling period, the media were removed, and the cells were washed twice with PBS at the same temperature as the chase incubation and then incubated with excess unlabeled met (10 mM)/cys (3 mM) in chase medium (for primary hepatocytes, Waymouth's MB 752/1 containing 1% streptomycin/penicillin, 1% L-glutamine, 0.2% BSA, and 0.1 nM insulin; for McA cells, high-glucose DMEM containing 0.5% FBS, 0.5% horse serum, 1% penicillin/streptomycin, and 1% L-glutamine).…”

“…To avoid this large effect on ROS, we used a second method: cooling the cells to 20°C, which allows trafficking into, but not out of, the Golgi (22,23). As a control, cooling OA-treated cells to 20°C blocked all secretion of 35 S-labeled apoB (see Fig. 5C).…”

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

“…That work first raised the possibility that VLDL was assembled in a two-step process. This scheme has been supported by studies from several laboratories (3)(4)(5), including our own (6). The two-step model includes the transformation of an "HDL-like" apoB-lipoprotein to a TG-enriched lipoprotein in the presence of adequate cell lipid stores (4,7).…”

“…Recent studies suggest that transformation may require either lateral diffusion of apoB to a specialized compartment of the ER or vesicular transport of apoB from one secretory compartment to another (4,13,14), but a review of the literature indicates that significant uncertainty exists regarding this issue (2,5,(15)(16)(17)(18)(19)(20). In the studies presented here, using pharmacological and subcellular fractionation approaches we demonstrate that the oleic acid (OA)-induced conversion of apoB100 LDL/HL particles to VLDL in McA RH7777 cells occurs in the ER and not in the Golgi.…”