Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus

J Biol Chem. 1997 Sep 19;272(38):23623-30. doi: 10.1074/jbc.272.38.23623.

Abstract

Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14 N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19 and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides. N-Glycosylation of Asn19 and Asn23 provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / metabolism*
  • Amines / chemistry
  • Animals
  • COS Cells
  • Cell Membrane / metabolism
  • Cytosol / metabolism
  • Drug Resistance, Multiple / genetics
  • Glycosylation
  • HeLa Cells
  • Humans
  • Multidrug Resistance-Associated Proteins
  • Mutagenesis, Site-Directed
  • Trypsin / chemistry

Substances

  • ATP-Binding Cassette Transporters
  • Amines
  • Multidrug Resistance-Associated Proteins
  • Trypsin