title: Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis.
abstract: 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 (125)I-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 Delta(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 (125)I-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 (125)I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 degrees 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.
authors.affiliation: Cardiovascular Genetics Laboratory, Division of Cardiology, McGill University Health Centre/Royal Victoria Hospital, Montréal, Québec H3A 1A1, Canada.
authors.forename: Houssein Hajj
authors.initials: HH
authors.lastname: Hassan
authors.forename: Dana
authors.initials: D
authors.lastname: Bailey
authors.forename: Dong-Young Donna
authors.initials: DY
authors.lastname: Lee
authors.forename: Iulia
authors.initials: I
authors.lastname: Iatan
authors.forename: Anouar
authors.initials: A
authors.lastname: Hafiane
authors.forename: Isabelle
authors.initials: I
authors.lastname: Ruel
authors.forename: Larbi
authors.initials: L
authors.lastname: Krimbou
authors.forename: Jacques
authors.initials: J
authors.lastname: Genest
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D017434:Protein Structure, Tertiary
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL