Topology Mapping of the Amino-terminal Half of Multidrug Resistance-associated Protein by Epitope Insertion and Immunofluorescence

Christina Kast,Philippe Gros

doi:10.1074/jbc.272.42.26479

Abstract

The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2. Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters. The topology of the membrane-associated regions of MRP remains largely unknown and was investigated. Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance. The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells. Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance. Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location. Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function. These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain. Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.

Highlights

Membrane Topology of multidrug resistance-associated protein (MRP)(HA epitope)c:YPYDVPDYAS a Nucleotide position in sequence of MRP published by Cole et al [10; 55]. b Amino acid residue immediately preceeding the site of insertion of the influenza virus hemagglutinin A (HA) epitope. c Influenza virus HA epitope sequence
§ Supported by a career award from the Medical Research Council of Canada and is an International Research Scholar of the Howard Hughes Medical Institute
Construction and Expression of Epitope-tagged Mutant multidrug resistance-associated protein (MRP)—Combined hydropathy analysis of MRP and other ATP-binding cassette (ABC) transporters suggests a protein with two hydrophilic ATPbinding domains, two membrane-associated regions in the amino-terminal half of the protein, and one membrane-associated region in the COOH-terminal half of the protein delineated by residues ϳ970 and 1260 (Fig. 1A)

Summary

Membrane Topology of MRP

(HA epitope)c:YPYDVPDYAS a Nucleotide position in sequence of MRP published by Cole et al [10; 55]. b Amino acid residue immediately preceeding the site of insertion of the influenza virus HA epitope. c Influenza virus HA epitope sequence. We have used a biochemical approach to initiate topology mapping of the membrane-associated regions of MRP This method is based on the insertion of a 10-amino acid antigenic peptide (HA epitope from influenza virus) in predicted hydrophilic peaks from the TM regions of MRP [37]. These mutant proteins are expressed in HeLa cells to test their capacity to confer drug resistance, and the polarity of the epitope tag with respect to the plasma membrane (intra- versus extracellular) is determined by immunofluorescence in intact or permeabilized cells using a monoclonal antibody directed against the epitope tag. We report on the topology mapping of the two membrane-associated regions from the aminoterminal half of MRP, including the unique domain characteristic of the MRP class of ABC transporters

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION