ABCC1 p.Leu1515Ala
| Predicted by SNAP2: | A: N (57%), C: N (72%), D: D (66%), E: D (59%), F: N (82%), G: D (59%), H: D (53%), I: N (97%), K: D (53%), M: N (97%), N: D (53%), P: D (63%), Q: N (53%), R: D (59%), S: N (53%), T: N (61%), V: N (82%), W: N (57%), Y: N (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] Identification and characterization of functionall... J Biol Chem. 2004 Dec 17;279(51):53571-83. Epub 2004 Sep 30. Westlake CJ, Payen L, Gao M, Cole SP, Deeley RG
Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region.
J Biol Chem. 2004 Dec 17;279(51):53571-83. Epub 2004 Sep 30., 2004-12-17 [PMID:15459206]
Abstract [show]
The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.
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No. Sentence Comment
227 The L1497A/I1498A/V1499A/L1500A mutation essentially inactivated the protein, and the double L1514A/L1515A mutation reduced transport by 80%.
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ABCC1 p.Leu1515Ala 15459206:227:100
status: NEW228 Similar to the MRP11-1527, the L1514A/L1515A mutant had a 4-fold increase in the Km for ATP and an approximate 2.5-fold decrease in the Vmax for LTC4 transport.
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ABCC1 p.Leu1515Ala 15459206:228:38
status: NEW256 Related structure COOH-terminal mutation Expression (relative to WT) LTC4 uptake (percentage of WT) Km Vmax -fold % M pmol mg-1 min-1 Helix C3 L1488A 1.2 48 Ϯ 2 I1491A 1.2 59 Ϯ 2 M1492A 1.4 89 Ϯ 3 Sheet C2 V1499A 1.0 94 Ϯ 6 L1500A 1.1 22 Ϯ 1 V1497A/I1498A/V1499A/L1500A 1.0 3.6 Ϯ 0.8 D1501A 1.0 90 Ϯ 5 Sheet C1 G1503A 0.8 100 Ϯ 4 I1505A 0.5 90 Ϯ 10 E1507A 0.8 108 Ϯ 6 G1509A 0.8 113 Ϯ 9 Helix C2 P1511A 1.0 82 Ϯ 6 L1514A/L1515A 1.2 18 Ϯ 2 623 84 L1514V/L1515V 1.0 75 Ϯ 1 L1514I/L1515I 1.0 94 Ϯ 2 Q1516A 1.2 94 Ϯ 3 R1518A 0.8 103 Ϯ 10 G1519A 1.1 100 Ϯ 10 L1520A 1.3 110 Ϯ 3 Helix C1 F1521A 0.8 38 Ϯ 1 F1521Y 1.0 105 Ϯ 2 F1521G 0.9 40 Ϯ 1 F1521Q 1.0 30 Ϯ 1 F1521E 1.0 22 Ϯ 1 Y1522A 1.2 90 Ϯ 5 S1523A 1.1 103 Ϯ 6 M1524A 1.0 130 Ϯ 2 M1524G 1.0 65 Ϯ 3 A1525G 1.0 100 Ϯ 4 M1524G/A1525G 1.0 21 Ϯ 3 733 68 K1526A 2.5 85 Ϯ 2 D1527A/G1529A 3.0 123 Ϯ 4 tation impaired nucleotide binding and trapping at NBD2 to an extent comparable with that resulting from removal of the COOH-terminal 4 amino acids (not shown).
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ABCC1 p.Leu1515Ala 15459206:256:501
status: NEW[hide] Role of the NH2-terminal membrane spanning domain ... Mol Biol Cell. 2005 May;16(5):2483-92. Epub 2005 Mar 16. Westlake CJ, Cole SP, Deeley RG
Role of the NH2-terminal membrane spanning domain of multidrug resistance protein 1/ABCC1 in protein processing and trafficking.
Mol Biol Cell. 2005 May;16(5):2483-92. Epub 2005 Mar 16., [PMID:15772158]
Abstract [show]
Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that independent of cell type, the truncated protein accumulates in early/recycling endosomes. Using a real-time internalization assay, we demonstrate that MSD0 is important for MRP1 retention in, or recycling to, the plasma membrane. We also show that MSD0 traffics independently to the cell surface and promotes membrane localization of the core-region of MRP1 when the two protein fragments are coexpressed. Finally, we demonstrate that MSD0 becomes essential for trafficking of MRP1 when the COOH-terminal region of the protein is mutated. These studies demonstrate that MSD0 and the COOH-terminal region contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated. These data explain apparent differences in the trafficking requirement for MSD0 and the COOH-terminal region of MRP1 compared with other ABCC proteins.
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No. Sentence Comment
162 MRP11-203-CFP restored plasma membrane localization both to MRP1204 -1501, MRP1204 -1527 and the double Leu to Ala mutation, MRP1204 -1531L1514A-L1515A, with resultant colocalization of the two fragments (Figure 7).
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ABCC1 p.Leu1515Ala 15772158:162:145
status: NEW