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J. Biol. Chem., Vol. 280, Issue 48, 39942-39949, December 2, 2005

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Intracellular Lipidation of Newly Synthesized Apolipoprotein A-I in Primary Murine Hepatocytes^*

From the Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Departments of Biochemistry, Microbiology, and Immunology, and Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

Hepatocytes, which are the main site of apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) expression, are also the main source of circulating high density lipoprotein. Here we have characterized the intracellular lipidation of newly synthesized apoA-I, in primary hepatocytes cultured with [³H]choline to label choline-phospholipids, low density lipoprotein-[³H]cholesterol to label the cell surface, or [³H]mevalonate to label de novo synthesized cholesterol. Phospholipidation of apoA-I is significant and most evident in endoplasmic reticulum (ER) and medial Golgi, both in the lumen and on the membrane fractions of the ER and medial Golgi. In the presence of cycloheximide, endogenous apoA-I is substantially phospholipidated intracellularly but acquires some additional lipid after export out of the cell. In cells labeled with low density lipoprotein-[³H]cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the secreted apoA-I rapidly accumulates cholesterol after secretion from the cell in the media. On the other hand, de novo synthesized cholesterol can lipidate apoA-I intracellularly. We also showed the interaction between apoA-I and ABCA1 in ER and Golgi fractions. In hepatocytes lacking ABCA1, lipidation by low density lipoprotein-cholesterol was significantly reduced at the plasma membrane, phospholipidation and lipidation by de novo synthesized sterols were both reduced in Golgi compartments, whereas ER lipidation remained mostly unchanged. Therefore, the early lipidation in ER is ABCA1 independent, but in contrast, the lipidation of apoA-I in Golgi and at the plasma membrane requires ABCA1. Thus, we demonstrated that apoA-I phospholipidation starts early in the ER and is partially dependent on ABCA1, with the bulk of lipidation by phospholipids and cholesterol occurring in the Golgi and at the plasma membrane, respectively. Finally, we showed that the previously reported association of newly synthesized apoA-I and apoB (Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2005) J. Biol. Chem. 280, 21612–21621) occurs after secretion at the cell surface.

Received for publication, July 15, 2005 , and in revised form, September 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

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