The minimum functional unit of human P-glycoprotein appears to be a monomer.

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.