Immunosuppressant target protein FKBP12 is required for P-glycoprotein function in yeast

The mammalian P-glycoprotein (Pgp) is a approximately 170-kDa membrane protein that mediates multidrug resistance in many chemotherapy-resistant tumors by effluxing toxic compounds from the cell. Pgp homologs are expressed in many organisms, from bacteria to yeast and mammals. Previous studies established a model system to analyze the function of murine, human, and Plasmodium falciparum Pgp by heterologous expression in the yeast Saccharomyces cerevisiae. However, such studies have been hampered by the inherent resistance of yeast cells to chemotherapeutic agents. We find that an erg6 mutation, which blocks the final synthetic step of the membrane sterol ergosterol, renders yeast sensitive to anthracyclines and dactinomycin, clinically relevant Pgp substrates. We demonstrate that expression of the murine mdr3 gene confers dactinomycin resistance in both the erg6 mutant yeast strain and in an erg6 rad52 DNA repair mutant yeast strain. Similarly, murine mdr3 expression confers resistance to the immunosuppressants cyclosporin A (CsA) and FK506 in a CsA-FK506-sensitive vph6 mutant yeast strain. CsA and FK506 are known to partially overcome Pgp-mediated drug resistance, suggesting the targets of these drugs might regulate Pgp function. We find that both murine mdr3 and the yeast Pgp homolog STE6 function in yeast mutants lacking the CsA target proteins cyclophilin A and calcineurin. In contrast, murine mdr3 function was severely compromised in yeast mutants lacking the FK506/rapamycin target protein FKBP12. Both wild-type FKBP12 and an F43Y FKBP12 mutant with reduced prolyl isomerase activity supported mdr3 function. Our results support the model that immunosuppressants reverse multidrug resistance by competing with other Pgp substrates but reveal that inhibition of FKBP12-dependent Pgp function may also contribute to reversal of multidrug resistance by FK506 and rapamycin.