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.
Highlights
The molecular interaction of apoA-I with the cell membrane ABCA1 transporter has important implications in reverse cholesterol transport
It is accepted that a retroendocytosis pathway plays an important role in the formation of nascent high density lipoprotein (HDL) particles, the structural determinants governing the dynamics of apoA-I lipidation at different cellular sites have not yet been elucidated
Development of a Quantitative Biotinylation Assay—To investigate the cellular compartmentalization and trafficking pattern of apoA-I in a cell culture model, we developed a quantitative assay based on cell surface biotinylation
Summary
Patient Selection—For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously [12]). To test whether the biotinylation of cell surface proteins was complete, BHK cells induced with mifepristone and incubated with 10 g/ml of 125I-apoA-I for 45 min at 4 °C were washed, and biotinylation was performed as described above. Biotinylated cells incubated with or without apoA-I at 4 °C throughout each assay and subjected to reducing agent were used as controls for the efficacy of biotin cleavage Under these conditions, more than 95% of biotinylated ABCA1 was cleaved by glutathione. Analysis of Nascent ApoA-I-containing Particles—125IApoA-I released to the medium at the time point (6 h) from mifepristone-induced BHK-ABCA1 cells was analyzed by twodimensional PAGGE, and the number of apoA-I molecules per particle was assessed by cross-linking with dithiobis(succinimidyl propionate), as described previously [20]. Two-tailed p values Ͻ0.05 were considered as significantly different
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