MEMBRANE TOPOLOGY OF THE MULTIDRUG-RESISTANCE PROTEIN (MRP) - A STUDYOF GLYCOSYLATION-SITE MUTANTS REVEALS AN EXTRACYTOSOLIC NH2 TERMINUS

Multidrug resistance protein, MRP, is a 190-kDa integral membrane phos phoglycoprotein that belongs to the ATP-binding cassette superfamily o f transport proteins and is capable of conferring resistance to multip le chemotherapeutic agents. Previous studies have indicated that MRP c onsists of two membrane spanning domains (MSD) each followed by a nucl eotide binding domain, plus an additional extremely hydrophobic NH2-te rminal MSD, Computer-assisted hydropathy analyses and multiple sequenc e alignments suggest several topological models for MRP. To aid in det ermining the topology most likely to be correct, we have identified wh ich of the 14 N-glycosylation sequons in this protein are utilized. Li mited proteolysis of MRP-enriched membranes and deglycosylation of int act MRP and its tryptic fragments with PNGase F was carried out follow ed by immunoblotting with antibodies known to react with specific regi ons of MRP. The results obtained indicated that the sequon at Asn(354) in the middle MSD is not utilized and suggested approximate sites of N-glycosylation, Subsequent site-directed mutagenesis studies establis hed that Asn(19) and Asn(23) in the NH2-terminal MSD and Asn(1006) in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides. N-Glycosylation of Asn(19) and Asn(23) pro vides the first direct experimental evidence that MRP has an extracyto solic NH2 terminus. This finding, together with those of previous stud ies, strongly suggests that the NH2-terminal MSD of MRP contains an od d number of transmembrane helices. These results may have important im plications for the further understanding of the interaction of drugs w ith MRP.