ABCMdb

ABCC1 p.Asn1156Gln

Predicted by SNAP2:	A: N (66%), C: N (53%), D: N (61%), E: N (61%), F: D (66%), G: N (93%), H: N (66%), I: D (71%), K: D (59%), L: D (53%), M: D (59%), P: D (59%), Q: N (78%), R: D (59%), S: N (97%), T: N (82%), V: D (66%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: N, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
66 The N19Q, N1006Q, and N1156Q mutations were generated using the TransformerTM site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) based on the method developed by Deng and Nickoloff (35). Login to comment
67 The templates for mutagenesis were prepared by cloning the BamHI fragment as above (for N19Q) and the XmaI fragment (MRP nucleotides 2337-4322) (for N1006Q and N1156Q) from pcDNAI-MRP1 into pGEM®-3Z (Promega, Madison, WI). Login to comment
68 Mutagenesis was then performed according to the manufacturer`s instructions using the ScaI/ StuI and SspI/EcoRV selection primers (for N19Q and N1006Q/N1156Q mutations, respectively), and the following sense mutagenic oligonucleotide primers: 5Ј-C TGG GAC TGG CAG GTC ACG TGG-3Ј (N19Q), 5Ј-C CCC ATC GTC CAG GGG ACT CAG G-3Ј (N1006Q), and 5Ј-C TAT TCC CAT TTC CAG GAG ACC TTG C-3Ј (N1156Q). Login to comment
69 The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEM௡-3Z as a template. Login to comment
172 We next examined expression of the MSD3 glycosylation acceptor site mutants MRP-(N1006Q) and MRP-(N1156Q). Login to comment
173 The MRP-(N1156Q) mutant co-migrated with the wild-type protein, whereas MRP-(N1006Q) displayed a greater electrophoretic mobility (Fig. 4A, lanes 2, 7 and 8). Login to comment
174 The C-1 fragments derived from wild-type MRP and MRP-(N1156Q) also co-migrated, and the C-1 fragment of MRP-(N1006Q) had the same electrophoretic mobility as the PNGaseF-treated wild-type C-1 fragment (Fig. 4C, lanes 1-4). 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
231 On the other hand, it was expected that the MRP-(N1156Q) mutant might be informative because this asparagine residue is predicted to be extracytoplasmic only in the MSD3 configurations with four transmembrane segments. Login to comment