The Minimum Functional Unit of Human P-glycoprotein Appears to be a Monomer

Tip W Loo,David M Clarke

doi:10.1074/jbc.271.44.27488

Abstract

Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His)10, even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drug-stimulated ATPase activity when the half-molecules NH2-terminal half-(His)10/COOH-terminal half-A52 or NH2-terminal half-A52/COOH-terminal half-(His)10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer.

Highlights

The mechanism by which P-glycoprotein is able to couple ATP hydrolysis to the efflux of a broad range of lipophilic compounds is unknown
To test the feasibility of such an approach, we tested for interactions between half-molecules of P-glycoprotein containing either a histidine tag or the epitope for monoclonal antibody A52 at the COOH terminus
Restoration of drug-stimulated ATPase activity was observed only when both half-molecules were expressed contemporaneously in insect cells. These results suggested that the two half-molecules could associate in vivo to form a functional complex. Since both of these half-molecules contained the epitope tag for monoclonal antibody A52 at their COOH terminus, it appeared that addition of residues to the terminus of either polypeptide did not interfere with their association or function

Summary

Introduction

The mechanism by which P-glycoprotein is able to couple ATP hydrolysis to the efflux of a broad range of lipophilic compounds is unknown. Our approach to identifying the minimum functional unit was to coexpress P-glycoproteins containing either a histidine tag or the epitope for monoclonal antibody A52 at the COOHterminal end of the molecule and test for associations using nickel-chelate chromatography.

Results

Conclusion