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J. Biol. Chem., Vol. 283, Issue 17, 11164-11175, April 25, 2008

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Quantitative Analysis of ABCA1-dependent Compartmentalization and Trafficking of Apolipoprotein A-I

IMPLICATIONS FOR DETERMINING CELLULAR KINETICS OF NASCENT HIGH DENSITY LIPOPROTEIN BIOGENESIS^*

From the Cardiovascular Genetics Laboratory, Division of Cardiology, McGill University Health Centre/Royal Victoria Hospital, Montréal, Québec H3A 1A1, Canada

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that ¹²⁵I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I (187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of ¹²⁵I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of ¹²⁵I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.

Received for publication, September 14, 2007 , and in revised form, January 23, 2008.

^* This work was supported by Grant MOP 15042 from the Canadian Institutes of Health Research and by the Heart and Stroke Foundation of Quebec. 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 Both authors contributed equally to this work.

² Holds the McGill University-Novartis Chair in Cardiology. To whom correspondence should be addressed: Division of Cardiology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Ave., West Montreal, Quebec H3A 1A1, Canada. Tel.: 514-934-1934 (ext. 34642); Fax: 514-982-0686; E-mail: jacques.genest{at}muhc.mcgill.ca.

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