Revisiting the Role of the Leucine Plug/Valve in the Human ABCG2 Multidrug Transporter.

In the human ABCG2 (ATP Binding Casette transporter G2/BCRP/MXR) multidrug transporter, a so-called "leucin plug/valve" (a.a. L554/L555) has been suggested to facilitate substrate exit and the coupling of drug transport to ATPase activity. In this work, we analyzed the effects of selected variants in this region by expressing these variants, both in mammalian and Sf9 insect cells. We found that, in mammalian cells, the L554A, L554F, L555F, and a combination of L554F/L555F variants of ABCG2 were functional, were processed to the plasma membrane, and exhibited substrate transport activity similar to the wild-type ABCG2, while the L555A and L554A/L555A mutants were poorly expressed and processed in mammalian cells. In Sf9 cells, all the variants were expressed at similar levels; still, the L555A and L554A/L555A variants lost all transport-related functions, while the L554F and L555F variants had reduced dye transport and altered substrate-stimulated ATPase activity. In molecular dynamics simulations, the mutant variants exhibited highly rearranged contacts in the central transmembrane helices; thus, alterations in folding, trafficking, and function can be expected to occur. Our current studies reinforce the importance of L554/L555 in ABCG2 folding and function, while they do not support the specific role of this region in selective substrate handling and show a general reduction in the coupling of drug transport to ATPase activity in the mutant versions.