ABCMdb

ABCC1 p.Cys148Ala

None has been submitted yet.

Publications

PMID: 12731862 [PubMed] Leslie EM et al: "Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)."

No. Sentence Comment

54 Mutagenesis was performed according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined and introduced or lost restriction sites are in italics) as follows: Cys43Ala (5'-G TGG GTG CCT GCT TTT TAC CTC TGG GCC-3'), Cys43Ser (5'-G TGG GTG CCT TCT TTT TAC CTC-3'), Cys49Ala (5'-C CTC TGG GCC GCA TTC CCC TTC TAC-3') (BsmI), Cys49Ser (5'-C CTC TGG GCC TCT TTC CCC TTC-3'), Cys85Ala (5'-G TGG ATC GTC GCG TGG GCA GAC C-3') (BstUI), Cys85Ser (5'-G TGG ATC GTC AGC TGG GCA GAC C-3'), Cys148Ala (5'-GTA GCC CTA GTG GCT GCC CTA GCC-3') (BglI), Cys148Ser (5'-GTA GCC CTA GTG TCT GCC CTA GCC-3'), Cys190Ala (5'-C GTC TTG TCC GCA TTC TCA GAT CGC-3') (BsmI), Cys190Ser (5'-C GTC TTG TCC TCT TTC TCA GAT CG-3'), Cys208Ala (5'-C CCT AAT CCC GCG CCA GAG TCC AG-3') (BstUI), Cys208Ser (5'-C CCT AAT CCC AGC CCA GAG TCC-3'), Cys265Ala (5'-GTA AAG AAC TGG AAG AAG GAA GCC GCG AAG ACT AGG AAG CAG-3') (BpiI), and Cys265Ser (5'- GG AAG AAG GAA TCC GCC AAG ACT AG-3') (BsmI). Login to comment
130 Table 1: Detection of Tryptic Fragments N1 and N2 of MRP1 in Membranes Prepared from HeLa Cells Stably Expressing CysfAla and CysfSer MRP1 Mutantsa trypsin:protein ratio (w:w)transfected HeLa cell line N1 detected N2 detected WT-MRP1 1:10 000 1:1000 C43A-MRP1 1:100 1:100 C43S-MRP1 1:10 000 1:1000 C49A-MRP1 1:250 1:100 C49S-MRP1 1:10 000 1:500 C85A-MRP1 1:10 000 1:1000 C85S-MRP1 1:1000 1:250 C148A-MRP1 1:250 1:250 C148S-MRP1 1:1000 1:500 C190A-MRP1 1:1000 1:1000 C190S-MRP1 1:1000 1:250 C208A-MRP1 1:10 000 1:250 C208S-MRP1 1:10 000 1:500 C265A-MRP1 1:250 1:10 C265S-MRP1 1:1000 1:250 a The data shown represent a summary of the limited trypsin digests shown in Figures 4 and 5. Login to comment
134 Trypsinolysis of the four remaining Ala-substituted Cys mutants (Cys148Ala, Cys49Ala, Cys43Ala, and Cys265Ala) showed that they were also quite resistant to cleavage by this enzyme. Login to comment
135 For example, the N1 and N2 fragments of Cys148Ala-MRP1 did not appear until a trypsin:protein ratio of 1:250 while the N1 and N2 fragments of Cys49Ala-MRP1 appeared at ratios of 1:250 and 1:100, respectively. The Cys43Ala-MRP1 mutant was highly resistant to trypsinolysis with the appearance of the N1 band not occurring until the trypsin:protein ratio was 1:100, the same ratio at which the N2 band appeared. Login to comment
153 Thus, a moderate (32-42%) decrease in E217 G uptake activity was observed for four of the five mutants (Cys49Ala-MRP1, Cys85Ala-MRP1, Cys148Ala-MRP1, and Cys265Ala-MRP1). Login to comment
203 The most significant decreases were observed in membranes from cells expressing the Cys43Ala, Cys49Ala, Cys148Ala, and Cys265Ala MRP1 mutants, suggesting that an alteration in the conformation of these proteins has occurred that decreases the accessibility of specific trypsin cleavage sites in CL3 (appearance of N2 fragment) and, in some cases, in the linker region of the protein between NBD1 and MSD3 (appearance of N1 fragment). Login to comment