Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein

Multidrug resistance of cancer cells is, at least in part, conferred by ove rexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters, P-gp actively extrudes chemothera peutic drugs from cells, thus reducing their efficacy, As a typical ABC tra nsporter, P-gp has four domains: two transmembrane domains, which form a pa thway through the membrane through which substrates are transported, and tw o hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substra te translocation, It has been proposed that the NBDs of ABC transporters, i ncluding the histidine permease of Salmonella typhimurium and the cystic fi brosis transmembrane conductance regulator, are accessible from the extrace llular surface of the cell, spanning the membrane directly or potentially c ontributing to the transmembrane pore. Such organization would have signifi cant implications for the transport mechanism, We determined to establish w hether the NBDs of P-gp are exposed extracellularly and which amino acids a re accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface, The implications for the structu re and mechanism of P-gp and other ABC transporters are discussed.