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Cysteine‐scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide‐binding domains of the multidrug resistance transporter P‐glycoprotein
Author(s) -
Blott Emma J.,
Higgins Christopher F.,
Linton Kenneth J.
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.23.6800
Subject(s) - atp binding cassette transporter , biology , transmembrane domain , biochemistry , transmembrane protein , cyclic nucleotide binding domain , mutagenesis , transporter , cysteine , p glycoprotein , atp hydrolysis , cystic fibrosis transmembrane conductance regulator , multidrug resistance associated proteins , permease , cell membrane , microbiology and biotechnology , multiple drug resistance , atpase , amino acid , nucleotide , mutation , cell , gene , enzyme , receptor , antibiotics
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P‐glycoprotein (P‐gp), a member of the ATP‐binding cassette (ABC) superfamily of active transporters. P‐gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P‐gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide‐binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P‐gp are exposed extracellularly and which amino acids are 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 structure and mechanism of P‐gp and other ABC transporters are discussed.