Title : Membrane topology of the
multidrug resistance protein (
MRP )
Abstract :
- A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus
- 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 2. phosphoglycoprotein, , Multidrug resistance protein, -, -
- 2. phosphoglycoprotein, , phosphoglycoprotein, -, -
- 5. N-glycosylation, , -, -, -
- 6. deglycosylation, , MRP, -, -
- 7. N-glycosylation, , -, -, sites
- 7. utilized, , -, -, Asn354
- 7. utilized, , -, -, sequon
- 8. modified, , -, N-linked oligosaccharides, Asn1006
- 8. modified, , -, N-linked oligosaccharides, Asn19 and Asn23
- 8. modified, , -, N-linked oligosaccharides, sites
- 9. N-Glycosylation, , -, -, Asn19 and Asn23
Output(Part-Of) (sent_index,
protein,
site):
- 5. protein, -
- 6. MRP, regions
- 8. MRP, sites
- 9. MRP, terminus
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 7. multidrug resistance protein, -, Asn354
- 8. multidrug resistance protein, N-linked oligosaccharides, Asn1006
- 8. multidrug resistance protein, N-linked oligosaccharides, Asn19 and Asn23
- 9. multidrug resistance protein, -, Asn19 and Asn23