The MDR-1 gene product, plasma membrane glycoprotein or P-glycoprotein (PGP), has been shown to confer drug resistance to cancer cells by acting as an energy-dependent drug-efflux pump. We have examined the endocytic traffic of PGP in human multidrug-resistant cells and tested whether the traffic and the steady-state intracellular localization of PGP can be experimentally modulated. Here we show that 1) under steady state approximately 70% of cellular PGP is on the surface whereas approximately 30% is intracellular, 2) surface PGP undergoes constitutive endocytosis and recycling, 3) endocytosis of PGP involves clathrin and adaptin complex 2-dependent mechanism, and 4) PGP cycles through a Rab5-responsive endosomal compartment. Biochemical (such as antibody crosslinking of PGP or treatment of cells with chloroquine) and molecular (such as overexpression of Rab5) treatments were used to modulate the endocytic/ recycling traffic of PGP. Such treatments resulted in the redistribution of PGP from the cell surface to intracellular compartments. Cells with such "mislocalized" PGP showed a decrease in multidrug resistance, suggesting that clinically relevant strategies can be attempted by modulating PGP's temporal and spatial distribution within cancer cells.

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Substrate (or solute)‐binding proteins (SBPs) selectively bind the target ligands and deliver them to the ATP‐binding cassette (ABC) transport system for their translocation. Irrespective of the different types of ligands, SBPs are structurally conserved. A wealth of structural details of SBPs bound to different types of ligands and the physiological basis of their import are available; however, the uptake mechanism of nucleotides is still deficient. In this study, we elucidated the structural details of an SBP endogenously bound to a novel ligand, a derivative of uridylyl‐3′‐5′‐phospho‐guanosine (U3G); thus, we named it a U3G‐binding protein (U3GBP). To the best of our knowledge, this is the first report of U3G (and a dinucleotide) binding to the SBP of ABC transport system, and thus, U3GBP is classified as a first member of subcluster D‐I SBPs. Thermodynamic data also suggest that U3GBP can bind phospholipid precursor sn‐glycerophosphocholine (GPC) at a site other than the active site. Moreover, a combination of mutagenic and structural information reveals that the protein U3GBP follows the well‐known ‘Venus Fly‐trap’ mechanism for dinucleotide binding. Databases Structural data are available in RCSB Protein Data Bank under the accession number(s) https://doi.org/10.2210/pdb7C0F/pdb, https://doi.org/10.2210/pdb7C0K/pdb, https://doi.org/10.2210/pdb7C0L/pdb, https://doi.org/10.2210/pdb7C0O/pdb, https://doi.org/10.2210/pdb7C0R/pdb, https://doi.org/10.2210/pdb7C0S/pdb, https://doi.org/10.2210/pdb7C0T/pdb, https://doi.org/10.2210/pdb7C0U/pdb, https://doi.org/10.2210/pdb7C0V/pdb, https://doi.org/10.2210/pdb7C0W/pdb, https://doi.org/10.2210/pdb7C0X/pdb, https://doi.org/10.2210/pdb7C0Y/pdb, https://doi.org/10.2210/pdb7C0Z/pdb, https://doi.org/10.2210/pdb7C14/pdb, https://doi.org/10.2210/pdb7C15/pdb, https://doi.org/10.2210/pdb7C16/pdb, https://doi.org/10.2210/pdb7C19/pdb, and https://doi.org/10.2210/pdb7C1B/pdb.

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