ABCMdb

ABCC1 p.Asn23Gln

None has been submitted yet.

Publications

PMID: 9295302 [PubMed] Hipfner DR et al: "Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus."

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
166 The MRP-(N19Q) and MRP-(N23Q) N-2 fragments are glycosylated because they have lower electrophoretic mobility than the deglycosylated 25-kDa N-2 fragment of wild-type MRP (compare with Fig. 4B, lane 1). Login to comment
168 To confirm that Asn19 and Asn23 are the only glycosylated sites in MSD1, we also produced an N19Q/N23Q MRP double mutant. 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