Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1838, Issue 3, Pages 756-765Publisher
ELSEVIER
DOI: 10.1016/j.bbamem.2013.11.002
Keywords
ATP-binding cassette; Multidrug resistance protein 1; Organic anion transporter; Plasma membrane trafficking; Protein misfolding; Chemical chaperones
Categories
Funding
- Canadian Institutes for Health Research [MOP-10519]
Ask authors/readers for more resources
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 (HEM) cells. Exposure of HEM 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 K613A 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 K-m. In contrast to K513A, K516A levels in HEM 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 K-m. 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. (C) 2013 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available