Abstract 828: Quantitative Analysis of Cellular Compartmentalization and Trafficking of ApoA-I: Implications for Nascent HDL Biogenesis
It is generally thought that the lipidation of apoA-I may occur inside the cell as a part of a retroendocytosis pathway. To investigate the molecular mechanisms underlying the genesis of HDL, we developed a cell surface biotinylation assay to quantitatively measure the association and dissociation of apoA-I from plasma membrane (PM) and intracellular compartments (ICC). 125I-apoA-I exhibited saturable binding that was found to occur in a concentration-dependent manner to both PM and ICC fractions in normal human fibroblasts treated with 22OH/9CRA. Tangier Disease fibroblasts showed no significant association of 125I-apoA-I with both PM and ICC. Kinetic analysis revealed that PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared to ICC (Bmax=0.21±0.01 ng apoA-I/μg cell protein vs. 0.10±0.01 ng/μg cell protein, respectively). In contrast, apoA-I was found to have similar affinity for PM and ICC (Kd=1.52±0.14 μg/mL vs. 1.68±0.42 μg/mL, respectively). To better understand the dynamics of the lipidation of apoA-I at different cellular sites, a pulse chase experiment was performed and the dissociation of 125I-apoA-I from PM and ICC was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from PM was 4-fold slower than that of ICC at 37°C (t1/2=126±24 min vs. 29±1.2 min, respectively), whereas 125I-apoA-I dissociation from PM and ICC was almost completely inhibited at 4°C. Interestingly, treatment of the cells with phospholipases PC-PLC, but not SMase increased the dissociation of apoA-I from PM, but not ICC. On the other hand, treatment of the cells with Brefeldin A and Monensin blocked cholesterol efflux and decreased significantly the dissociation of apoA-I specifically from ICC. Finally the majority of apoA-I released to the medium was found lipidated with particle diameters ranging from 90 Å to 170 Å. Altogether these data suggest that the lipidation of apoA-I occurs in two kinetically distinguishable compartments, the slow dissociation of apoA-I from a major lipidation site at the PM and the fast re-secretion of lipidated apoA-I from ICC that seems to be differentially modulated. The results of the present study have implications for current models of nascent HDL biogenesis.