ABCB1 p.Asn280Cys
| Predicted by SNAP2: | A: N (53%), C: N (61%), D: N (66%), E: D (53%), F: N (53%), G: N (57%), H: N (93%), I: D (59%), K: N (78%), L: N (53%), M: N (53%), P: D (53%), Q: N (61%), R: N (53%), S: N (72%), T: N (61%), V: D (53%), W: D (63%), Y: N (57%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: N, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Cysteine-scanning mutagenesis provides no evidence... EMBO J. 1999 Dec 1;18(23):6800-8. Blott EJ, Higgins CF, Linton KJ
Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.
EMBO J. 1999 Dec 1;18(23):6800-8., 1999-12-01 [PMID:10581253]
Abstract [show]
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.
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No. Sentence Comment
36 Two additional single-cysteine (SC) mutants (N280C and G324C), known to be located in an intracellular and extracellular loop of TMD1, respectively (Loo and Clarke, 1995), were also generated as controls.
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ABCB1 p.Asn280Cys 10581253:36:45
status: NEW38 P-gp-cys- and 21 of the 30 SC mutants (including the control mutants SC-N280C and SC-G324C) expressed full-length protein in both the lower molecular weight non-glycosylated and the higher molecular weight glycosylated forms (Figure 2).
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ABCB1 p.Asn280Cys 10581253:38:72
status: NEW66 To establish the validity of the assay, SC-P-gp mutants G324C (known to be exposed in an extracellular loop of TMD1) and N280C (known to be located in an intracellular loop of TMD1) were used as positive and negative controls, respectively.
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ABCB1 p.Asn280Cys 10581253:66:121
status: NEW67 In intact cells, the higher molecular weight, glycosylated P-gp-G324C was biotinylated while neither form of P-gp- N280C was biotinylated (Figure 4A), as expected.
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ABCB1 p.Asn280Cys 10581253:67:115
status: NEW68 To demonstrate that P-gp-N280C was expressed and could be labelled by BM, the same assay was carried out in the presence of saponin, a gentle membrane permeabilizing agent.
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ABCB1 p.Asn280Cys 10581253:68:25
status: NEW69 In this case, both the mature and immature (nonglycosylated) forms of G324C and N280C P-gp were labelled equivalently (Figure 4B): immature P-gp is likely to be confined to intracellular membranes and so accessible to BM only after permeabilization of the cell.
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ABCB1 p.Asn280Cys 10581253:69:80
status: NEW190 Oligonucleotides used for site-directed mutagenesis pSC-name Diagnostic restriction site Mutagenic oligonucleotide sequence 5Ј-3Ј N280C ϩAvaI GGTACAACAAATGTCTCGAGGAAGCTAAAAG G324C -AflIII CCTTGGTCTTATCATGTGAATATTCTATTGG S403C ϩPstI CTTCAGTTACCCCTGCAGAAAAGAAGTTAAG S419C ϩEco57I GAACCTGAAAGTGCAGTGTGGGCAGACG Q456C ϩBstEI GTTGATGGATGCGATATCCGGACCATAAATG F465C ϩBanI ATGTAAGGTGCCTACGGGAA I469C ϩNsiI CTACGGGAATGCATTGGTGT S474C ϩHpaII GGTGTCGTGTGTCAGGAACCGGTATTGTTT T482C ϩBsrGI GTATTGTTTGCCTGTACAATAGCTGAAAAC I488C ϩEclXI GCTGAAAACTGTCGCTACGGCCGTGAAAATG Y490C none ACATTCGCTGTGGCCGTGA V495C ϩBsrGI GGCCGTGAAAATTGTACAATGGATGAGATTG D498C -NcoI GTCACCATGTGTGAGATTGAG I500C ϩBsmI GTCACCATGGATGAATGCGAGAAAGCTGTC K502C ϩBsmI GGATGAGATTGAATGCGCTGTCAAGGAAG N508C ϩFspI GTCAAGGAAGCATGCGCATATGACTTTATC D511C ϩAflIII GGAAGCCAACGCGTATTGCTTTATCATG K515C ϩAflIII GAAGCCAACGCGTATGACTTTATCATGTGCCTGCCTCAT K519C ϩStyI GAAACTGCCTCATTGCTTTGACACCTTGGTTGGAGAGAGAG A529C ϩBanI GAGAGAGGGTGCCAGTTGAG K536C ϩBlpI GAGAGAGAGGCGCCCAGTTGAGTGGTGGGCAGTGCCAGAGGATCG R547C ϩBssSI GCACGTGCCCTCGTGTGCAACCCCAAG P549C ϩSspI TGGTTCGCAACTGCAAAATATTCCTGCTGGA L554C ϩSspI CGCAACCCCAAAATATTGCTGTGCGATGAGGCCACG S565C ϩBsmI GACACAGAATGCGAAGCAG V569C ϩBsmI GACACAGAATGCGAAGCAGTGTGTCAGGTGG K578C ϩBsiEI GATAAGGCCAGATGCGGCCGGACCACC R580C -MslI GCCACAAAAGGTTGCACGACCATTGTGATA T581C ϩApaLI GAAAAGGTCGGTGCACCATTGTG E638C ϩEclXI GAAGTTGAATTATGCAATGCGGCCGATGAATC 'ϩ` represents the introduction of a new restriction endonuclease site; '-` represents the removal of an existing site.
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ABCB1 p.Asn280Cys 10581253:190:142
status: NEW