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J. Biol. Chem., Vol. 277, Issue 34, 31187-31200, August 23, 2002
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From the 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.
Intracellular Assembly of Very Low Density Lipoproteins
Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777
Cells*
,,
,**
Lipoprotein & Atherosclerosis
Group, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7,
Canada, the § Department of Chemistry, University of
Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, the Canadian
Food Inspection Agency, National Centre for Foreign Animal Disease,
Winnipeg, Manitoba R3E 3M4, Canada, the ** Department of
Pathology & Laboratory Medicine, Department of Biochemistry,
Microbiology & Molecular Immunology, University of Ottawa, Ottawa,
Ontario K1Y 4W7, Canada, and the ¶ Department of Biochemistry,
Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157
*
This work is supported by operating grants from the Canadian
Institute of Health Research (to Z. Y.), the Heart and Stroke Foundation of Ontario (to Z. Y.), and the Natural Sciences and Engineering Research Council of Canada (to J. C. J.),
National Institute of Health/National Cancer Institute Grant R01CA79670 (to Z. C.), and Signal Transduction and Cellular Function Training Grant CA-09422 (to C. J. D.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Scientist of Canadian Institutes of Health Research. To whom
correspondence should be addressed. Tel.: 613-798-5555 (ext. 18711); Fax: 613-761-5281; E-mail: zyao@ottawaheart.ca.
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