Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.
Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1.
Division of Cancer Biology and Genetics, Queen's University, Kingston, ON K7L 3N6, Canada; Department of Pathology & Molecular Medicine, Queen's University, Kingston, ON K7L 3N6, Canada.
Surtaj H
SH
Iram
Division of Cancer Biology and Genetics, Queen's University, Kingston, ON K7L 3N6, Canada; Department of Pathology & Molecular Medicine, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: spc.cole@queensu.ca.
Susan P C
SP
Cole
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D018682:GABA Agents / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects*; D017510:Protein Folding; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018682:GABA Agents; D005990:Glycerol; D008239:Lysine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011092:Polyethylene Glycols; D013501:Surface-Active Agents; D005680:gamma-Aminobutyric Acid; C421109:multidrug resistance-associated protein 1
4-PBA; 4-phenylbutyric acid; ABC; ATP-binding cassette; CL; Chemical chaperones; LTC(4); MRP1; MSD; Multidrug resistance protein 1; NBD; Organic anion transporter; PEG; Plasma membrane trafficking; Protein misfolding; TBS; TM; TMAO; Tris-buffered saline; cytoplasmic loop; leukotriene C(4); membrane spanning domain; multidrug resistance protein 1; nucleotide binding domain; polyethylene glycol; transmembrane; trimethylamine-N-oxide