ABCC1 p.Gly663Val
| Predicted by SNAP2: | A: N (61%), C: D (59%), D: N (82%), E: N (61%), F: D (75%), H: N (53%), I: D (75%), K: N (66%), L: D (75%), M: D (71%), N: N (93%), P: D (71%), Q: D (59%), R: N (61%), S: N (93%), T: D (63%), V: D (66%), W: D (85%), Y: D (75%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D, |
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[hide] Domain interactions in the yeast ATP binding casse... J Bacteriol. 2001 Aug;183(16):4761-70. Falcon-Perez JM, Martinez-Burgos M, Molano J, Mazon MJ, Eraso P
Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains.
J Bacteriol. 2001 Aug;183(16):4761-70., [PMID:11466279]
Abstract [show]
The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.
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No. Sentence Comment
55 To determine whether the reversion occurred at the site of the original mutation, the DNA regions that include the primary mutation were sequenced in the plasmids rescued from revertants of G663V, G756D, and G1306E mutations.
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ABCC1 p.Gly663Val 11466279:55:190
status: NEW101 For the intragenic suppressor analysis of mutations localized in the NBDs, we selected G663V, G756D, D777N, G1306E, and G1311R changes (Fig. 1), all of which affect residues in highly conserved motifs.
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ABCC1 p.Gly663Val 11466279:101:87
status: NEW108 Mutagenesis of plasmids containing the G663V, G756D, D777N, and G1306E mutations resulted in transformants that grew in the presence of Cd2ϩ , whereas mutagenesis of a plasmid containing the G1311R allele did not (Table 1).
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ABCC1 p.Gly663Val 11466279:108:39
status: NEW111 All revertants of G663V, G756D, and G1306E mutants were full revertants of the initial mutation, and 30 out of 83 revertants isolated for the D777N mutant were due to a second-site suppressor mutation.
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ABCC1 p.Gly663Val 11466279:111:18
status: NEW130 of revertants Full revertants Second-site suppressor mutants G663V Episomal Mutator strain 16 ϫ 104 18 0 G756D Centromeric Chemical 5 ϫ 104 0 0 Episomal Mutator strain 5 ϫ 104 2 0 Chemical 3 ϫ 104 15 0 D777N Episomal Mutator strain 16 ϫ 104 53 30 G1306E Centromeric Chemical 3 ϫ 104 1 0 Mutator strain 5 ϫ 104 4 0 G1311R Centromeric Chemical 3 ϫ 104 0 0 Episomal Chemical 3 ϫ 104 0 0 TABLE 2.
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ABCC1 p.Gly663Val 11466279:130:61
status: NEW204 We performed a revertant analysis of five mutations located in the NBDs of Ycf1p, namely G663V, G756D, D777N, G1306E, and G1311R.
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ABCC1 p.Gly663Val 11466279:204:89
status: NEW207 We were unable to identify any intragenic suppressors of the other four alleles, G663V, G756D, G1306E, and G1311R.
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ABCC1 p.Gly663Val 11466279:207:81
status: NEW212 Moreover, the NBD1 mutations G663V and G756D were not suppressed when combined with some of the suppressors isolated for D777N (see below).
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ABCC1 p.Gly663Val 11466279:212:29
status: NEW230 The specificity of the suppression is further supported by the fact that neither the W1225C nor R1415G mutations were able to suppress the defective growth or Ycf1p transport function of the other NBD1 primary mutations, namely G663V and G756D (data not shown).
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ABCC1 p.Gly663Val 11466279:230:228
status: NEW