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      Vacuolar ATPase Activity Required for ABCA1 Mediated Cholesterol Efflux

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          Abstract

          Objective

          We have shown that ABCA1 mediates unfolding of the apoA1 N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the vacuolar ATPase (V-ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1 mediated cholesterol efflux.

          Approach and Results

          We found that V-ATPase inhibitors dose dependently decreased ABCA1 mediated cholesterol efflux to apoA1 in baby hamster kidney (BHK) cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V 0C inhibited ABCA1 mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V 0A1 and V 1E1 subunits of V-ATPase. We generated a FITC/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose FITC/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in BHK cells led to acidification of the cell associated apoA1 pH indicator, compared to control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1, dose dependently inhibited the apoA1 pH shift in ABCA1 expressing cells, without affecting the levels of cell associated apoA1. However, we were not able to detect ABCA1 mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes.

          Conclusions

          Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1 mediated HDL biogenesis and lipid efflux.

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          Author and article information

          Journal
          9505803
          8623
          Arterioscler Thromb Vasc Biol
          Arterioscler. Thromb. Vasc. Biol.
          Arteriosclerosis, thrombosis, and vascular biology
          1079-5642
          1524-4636
          10 September 2018
          November 2018
          01 November 2019
          : 38
          : 11
          : 2615-2625
          Affiliations
          [1 ]Department of Cellular and Molecular Medicine Cleveland Clinic, Cleveland OH 44195
          [2 ]Department of Cellular Cardiovascular Medicine, Cleveland Clinic, Cleveland OH 44195
          Author notes
          Address correspondence to: Jonathan D. Smith, NC10, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, Phone: 216-444-2248, FAX: 216-444-9404, smithj4@ 123456ccf.org .
          Article
          PMC6209108 PMC6209108 6209108 nihpa1506150
          10.1161/ATVBAHA.118.311814
          6209108
          30354238
          de8dd107-41ba-47ae-817b-9573933fb61d
          History
          Categories
          Article

          Lipids and Cholesterol,ABCA1,Cell Biology/Structural Biology,apoA1 lipidation,vacuolar ATPase,apoA1 acidification

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