Mistargeted MRPΔF728 mutant is rescued by intracellular GSH

Abstract 

The most common cystic fibrosis-causing mutation is the deletion of the widely conserved phenylalanine 508 (ΔF508) of CFTR. The mutant is unable to fold correctly and to transit to the plasma membrane. MRP1 belongs to the same subfamily of ABC proteins as CFTR and confers resistance to a wide range of chemotherapeutic drugs. By analogy, phenylalanine 728 was deleted in MRP1. Our results shown that MRPΔF728 is correctly targeted to the plasma membrane, actively transports doxorubicin (DOX) and vincristine (VCR) and shares a structure identical to MRP1. Intracellular GSH depletion however results in a mistargeted mutant that is retained into the cytoplasm, while in the same conditions wild-type MRP1 is correctly routed to the plasma membrane. The GSH-protein complex could adopt a stable conformation protected against proteolytic degradation and correctly targeted to the plasma membrane.

Abbreviations: ABC, ATP binding cassette, ATP, adenosine-5′-triphosphate, ATR, attenuated total reflection, BSO, DL-buthionine-[S,R]-sulfoximine, CFTR, cystic fibrosis conductance regulator, DMSO, dimethyl sulfoxide, DOX, doxorubicin, FTIR, Fourier transform infrared spectroscopy, GSH, glutathione, H/D, hydrogen/deuterium, HEK, human embryonic kidney, LTC₄, leukotrienes C₄, MDR, multidrug resistance, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MRP, multidrug resistance protein, NBD, nucleotide binding domain, PCR, polymerase chain reaction, Pgp, P-glycoprotein, TMD, transmembrane domain, VCR, vincristine

Keywords: ABC, MRP1, MRPΔF728, Infrared spectroscopy, Membrane targeting, Fluorescence microscopy