ABCMdb

PMID: 9295302

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., [PubMed]

Sentences

No. Mutations Sentence Comment

61 ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln The N23Q and N71Q mutations were generated by a modification of the PCR-based megaprimer method (34). Login to comment 62 ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln The N23Q and N71Q mutations were generated by a modification of the PCR-based megaprimer method (34). Login to comment 63 ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln 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 64 ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln 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 66 ABCC1 p.Asn19Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEM¾1;-3Z as a template. Login to comment 70 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEMt-3Z as a template. Login to comment 72 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln The N19Q/N23Q/N1006Q triple mutant was prepared by cloning the BamHI fragment containing the N19Q/N23Q mutation into pcDNAI-MRP1/N1006Q. Login to comment 164 ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln 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 172 ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln We next examined expression of the MSD3 glycosylation acceptor site mutants MRP-(N1006Q) and MRP-(N1156Q). Login to comment 173 ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn19Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn23Gln ABCC1 p.Asn71Gln ABCC1 p.Asn71Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1156Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln ABCC1 p.Asn1006Gln 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 ABCC1 p.Asn1156Gln 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