ABCC1 p.Gly756Asp
| Predicted by SNAP2: | A: D (59%), C: D (63%), D: D (75%), E: D (75%), F: D (80%), H: D (63%), I: D (75%), K: D (71%), L: D (75%), M: D (75%), N: D (63%), P: D (71%), Q: D (66%), R: N (66%), S: D (53%), T: D (71%), V: D (71%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, 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.Gly756Asp 11466279:55:197
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.Gly756Asp 11466279:101:94
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.Gly756Asp 11466279:108:46
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.Gly756Asp 11466279:111:25
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.Gly756Asp 11466279:130:111
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.Gly756Asp 11466279:204:96
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.Gly756Asp 11466279:207:88
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.Gly756Asp 11466279:212:39
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.Gly756Asp 11466279:230:238
status: NEW[hide] Mutational analysis of the Saccharomyces cerevisia... Mol Microbiol. 1997 Aug;25(4):683-94. Wemmie JA, Moye-Rowley WS
Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p.
Mol Microbiol. 1997 Aug;25(4):683-94., [PMID:9379898]
Abstract [show]
Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.
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No. Sentence Comment
133 These mutants corresponded to CFTR alterations known to be associated with cystic fibrosis (G551D and G551S in CFTR, G756D and G756S in Ycf1p) as well as lesions that either disturb normal function (K464M in CFTR, K669M in Ycf1p) or act to suppress the phenotype of ⌬F508 CFTR (R553Q and R553M in CFTR, K758Q and K758M in CFTR).
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ABCC1 p.Gly756Asp 9379898:133:117
status: NEW135 K669M, G756S and G756D were unable to complement the cadmium hypersensitivity of the ⌬ycf1 strain. Western blot analysis of these mutant Ycf1p derivatives indicated that all these proteins were produced at the same level in the cell.
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ABCC1 p.Gly756Asp 9379898:135:17
status: NEW237 The three mutant Ycf1p derivatives (⌬F713, G756D and G756S) that correspond to known CF-causing alleles of CFTR (⌬F508, G551D and G551S) all produce a Ycf1p mutant that exhibits a defect analogous to its CFTR counterpart.
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ABCC1 p.Gly756Asp 9379898:237:50
status: NEW281 The 2 m plasmids carrying the indicated mutant forms of YCF1 and the relevant mutagenic PCR primers are listed below: pJAW100 (G756S), 5Ј-ATC TCC TTA TCT GGA TCC CAA AAA GCT CGT TTG-3Ј; pJAW88 (G756D), 5Ј-ATC TCC TTA TCT GGA GAC CAA AAA GCT CGT TTG-3Ј; pJAW86 (K669M), 5Ј- AAA GTT GGC AGT GGT ATG ACA GCT CTA TT-3Ј; pJAW98 (K758R), 5Ј-TTA TCT GGA GGA CAA CGG GCC CGT TTG TCT TTA-3Ј; pJAW108 (K758M), 5Ј-AGA CAA ACG AGC CAT TTG TCC TCC AGA TAA GGA TAT CCC TTT C-3Ј; pJAW109 (K758Q), 5Ј-AGA CAA ACG AGC TTG TTG TCC TCC AGA TAA GCT TAT CCC TTT C-3Ј.
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ABCC1 p.Gly756Asp 9379898:281:214
status: NEW[hide] Long-range coupling between the extracellular gate... FASEB J. 2015 Nov 25. pii: fj.15-278382. Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PMID:26606940]
Abstract [show]
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that: 1) all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy and 2) highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman, J. L., IV, Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
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No. Sentence Comment
70 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15).
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ABCC1 p.Gly756Asp 26606940:70:322
status: NEW88 For reference, we also introduced each phenylalanine mutation into the WT Ycf1p background and into an NBD1 signature sequence mutant that is expected to have negligible ATPase activity (G756D in Ycf1p) (16, 37, 38).
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ABCC1 p.Gly756Asp 26606940:88:187
status: NEW108 Mutating the conserved extracellular F565 residue partially restores the function of ATP binding mutants of Ycf1p (Y1281G and D777N), but not of a signature sequence mutant (G756D).
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ABCC1 p.Gly756Asp 26606940:108:174
status: NEW110 The WT and ATP binding mutations, Y1281G (top panel) and D777N (middle), or the signature sequence mutation, G756D (bottom), are indicated along with the F565 substitutions, introduced alone or in combination.
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ABCC1 p.Gly756Asp 26606940:110:109
status: NEWX
ABCC1 p.Gly756Asp 26606940:110:270
status: NEW111 The vertical rectangular box indicates spot cultures analyzed in B and C. B) Restoration of Ycf1p function is observed as differential growth associated with F565 substitutions in the context of the Y1281G (top) and D777N (middle) loss of function mutations but not the G756D signature sequence mutation (bottom).
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ABCC1 p.Gly756Asp 26606940:111:270
status: NEW114 C) Functional comparison between each single mutant, Ycf1p-Y1281G (top), Ycf1p-D777N (middle) or Ycf1p-G756D (bottom), and the respective A, S, and L substitutions for F565.
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ABCC1 p.Gly756Asp 26606940:114:103
status: NEW69 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15).
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ABCC1 p.Gly756Asp 26606940:69:322
status: NEW87 For reference, we also introduced each phenylalanine mutation into the WT Ycf1p background and into an NBD1 signature sequence mutant that is expected to have negligible ATPase activity (G756D in Ycf1p) (16, 37, 38).
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ABCC1 p.Gly756Asp 26606940:87:187
status: NEW107 Mutating the conserved extracellular F565 residue partially restores the function of ATP binding mutants of Ycf1p (Y1281G and D777N), but not of a signature sequence mutant (G756D).
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ABCC1 p.Gly756Asp 26606940:107:174
status: NEW109 The WT and ATP binding mutations, Y1281G (top panel) and D777N (middle), or the signature sequence mutation, G756D (bottom), are indicated along with the F565 substitutions, introduced alone or in combination.
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ABCC1 p.Gly756Asp 26606940:109:109
status: NEW113 C) Functional comparison between each single mutant, Ycf1p-Y1281G (top), Ycf1p-D777N (middle) or Ycf1p-G756D (bottom), and the respective A, S, and L substitutions for F565.
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ABCC1 p.Gly756Asp 26606940:113:103
status: NEW