ABCMdb

ABCC1 p.Asn71Gln

Predicted by SNAP2:	A: N (66%), C: N (82%), D: D (53%), E: D (59%), F: N (78%), G: N (66%), H: N (87%), I: N (66%), K: N (53%), L: N (72%), M: N (57%), P: N (57%), Q: N (72%), R: N (61%), S: N (82%), T: N (78%), V: N (66%), W: N (66%), Y: N (78%),
Predicted by PROVEAN:	A: D, C: D, D: N, E: D, F: D, G: D, H: N, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D,

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Publications
[hide] Membrane topology of the multidrug resistance prot... J Biol Chem. 1997 Sep 19;272(38):23623-30. Hipfner DR, Almquist KC, Leslie EM, Gerlach JH, Grant CE, Deeley RG, Cole SP
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., [PMID:9295302]

Abstract [show]
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.

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No. Sentence Comment
61 The N23Q and N71Q mutations were generated by a modification of the PCR-based megaprimer method (34). Login to comment
63 The first PCR was carried out on the pBluescript construct containing the BamHI fragment cloned in the EcoRI to HindIII orientation (5Ј 3 3Ј) using the M13 primer and either the N23Q (5Ј-C ACG TGG CAA ACC AGC AAC CCC GAC T-3Ј) or the N71Q (5Ј-A CCT CTC CAG AAA ACC AAA ACT GCC T-3Ј) mutagenic primer (substituted nucleotide positions underlined). Login to comment
164 Introduction of the individual N19Q, N23Q, and N71Q mutations in MSD1 did not result in a detectable alteration of the electrophoretic mobility of the intact proteins (Fig. 4A, lanes 3-5). Login to comment
165 However, after digestion with trypsin, the N-2 fragments of wild-type MRP and MRP-(N71Q) co-migrated at 43-60 kDa, whereas the N-2 fragments of MRP-(N19Q) and MRP-(N23Q) migrated slightly faster at approximately 38-50 kDa (Fig. 4B, lanes 2-5). Login to comment
169 MRP-(N19Q/ N23Q) migrated faster than wild-type MRP and the N19Q, N23Q, and N71Q single mutants (Fig. 4A, compare lane 6 with lanes 2-5). Login to comment
178 A, crude membrane proteins from Cos-1 cells expressing wild-type MRP (WT) treated with (ϩ) or without (-) PNGase F, the single glycosylation site mutant MRP constructs N19Q, N23Q, N71Q, N1006Q, and N1156Q, the double mutant N19Q/N23Q, and the triple mutant N19Q/N23Q/N1006Q were separated by SDS-polyacrylamide gel electrophoresis on 5% gels and immunoblotted with mAb QCRL-1. Login to comment
62 The N23Q and N71Q mutations were generated by a modification of the PCR-based megaprimer method (34). Login to comment
64 The first PCR was carried out on the pBluescript construct containing the BamHI fragment cloned in the EcoRI to HindIII orientation (59 3 39) using the M13 primer and either the N23Q (59-C ACG TGG CAA ACC AGC AAC CCC GAC T-39) or the N71Q (59-A CCT CTC CAG AAA ACC AAA ACT GCC T-39) mutagenic primer (substituted nucleotide positions underlined). Login to comment