Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase
Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation.
Methods and Results: We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH < 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37oC led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21oC or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V1 domain and an embedded V0 domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V0A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V0A1 subunit.
Conclusion: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.
Author Disclosures: S. Wang: None. G. Brubaker: None. J.D. Smith: None.
- © 2015 by American Heart Association, Inc.