ABCMdb

ABCC1 p.Phe565Leu

Predicted by SNAP2:	A: D (66%), C: D (59%), D: D (85%), E: D (80%), G: D (75%), H: D (80%), I: D (66%), K: D (80%), L: N (61%), M: D (66%), N: D (75%), P: D (91%), Q: D (80%), R: D (85%), S: D (75%), T: D (75%), V: D (63%), W: D (75%), Y: D (53%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: 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: N,

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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
10 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. Login to comment
65 Once the second-site mutation was identified, a 1.3-kb BsmI-StuI fragment for V543I, F565L, and S674L suppressors or a 1.8-kb NdeI-SalI fragment for the remainder of the suppressors was excised from the revertant and exchanged with the corresponding fragment in pRS425- ycf1D777N-HA. Login to comment
68 The plasmids containing both the original D777N and the second-site mutations were digested with BsmI and NcoI for V543I, F565L, and S674L suppressors or with NdeI and SalI for the remaining suppressors. Login to comment
132 Codon change(s)a Amino acid change(s)b 1 TTC3CTC F565L 2 -c - 3 GCC3GTC A1021V 4 GTT3ATT V5431 5 GGT3GAT G1207D 6 GCC3ACC A1021T 7 GCC3ACC A1021T 8 TTA3CTA, TGG3TGC L677L, W1225C 9 - - 10 TGG3TGC W1225C 11 GCC3GTC A1021V 12 TGG3TGC W1225C 13 GCC3GTC A1021V 14 CAG3CGG, TTA3TTG Q1107R, L1418L 15 TCA3TTA S674L 16 TGG3TGT, ACT3GCT W1225C, T1454A 17 TGG3TGT W1225C 18 GGT3GAT G1207D 19 - - 20 TGG3TGC W1225C 21 GCC3ACC A1021T 22 - - 23 GCA3GTA A1003V 24 GGT3AGT G1207S 25 AGA3GGA R1415G 26 GGT3AGT G1207S 27 TCA3TTA S1212L 28 GCC3GTC A1021V 29 AAC3GAC N1027D 30 TGG3TGC W1225C a The nucleotide changes are underlined. Login to comment
138 Two mutations (V543I and F565L) were localized in TMD1, nine substitutions (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) were found within TMD2, one mutation (S674L) was localized in NBD1, and another (R1415G) was in NBD2. Login to comment
149 Although the original mutant failed to grow on 1.5 mM diamide, 11 suppressed mutants grew at this concentration, of which 5 grew at an even higher concentration (V543I, F565L, Q1107R, G1207D, G1207S). Login to comment
154 On the other hand, among the group of mutants that corrected the Cd2ϩ defect to a similar extent (V543I, F565L, A1003V, A1021T, A1021V, Q1107R, G1207D, G1207S, and S1212L), only five (V543I, F565L, Q1107R, G1207D, and G1207S) were able to grow on 2 mM diamide (Fig. 3). Login to comment
175 The highest activity was observed in mutants N1027D (147%) and G1207S (138%), whereas mutants F565L (33%) and G1207D (47%) had the lowest. Login to comment
187 Second, mutants V543I, F565L, and Q1107R grew on diamide medium as much as the wild-type control, whereas growth on Cd2ϩ was clearly reduced (MICs ranging from 37 to 60% of that for the control). Login to comment
191 In addition, it revealed a group of suppressor mutants, V543I, F565L, Q1107R, G1207S, and W1225C, with a switch in their resistance profile indicating that Val543, Phe565, Gln1107, Gly1207, and Trp1225 are involved in determination of substrate specificity. Login to comment
197 Effect of the D777N suppressor mutations on the kinetic parameters of LTC4 uptake in vacuolar membrane vesicles Mutant Km (ATP)a Vmax a Wild type 0.05 Ϯ 0.01 4.40 Ϯ 0.31 D777N 1.42 Ϯ 0.18 1.69 Ϯ 0.24 D777N/V543I 1.09 Ϯ 0.28 1.15 Ϯ 0.04 D777N/F565L 1.44 Ϯ 0.30 0.58 Ϯ 0.04 D777N/S674L 1.37 Ϯ 0.26 1.21 Ϯ 0.05 D777N/A1003V 1.55 Ϯ 0.46 1.64 Ϯ 0.13 D777N/A1021T 1.14 Ϯ 0.20 1.28 Ϯ 0.12 D777N/A1021V 1.71 Ϯ 0.39 1.47 Ϯ 0.15 D777N/N1027D 1.16 Ϯ 0.18 2.49 Ϯ 0.34 D777N/Q1107R 1.07 Ϯ 0.09 1.48 Ϯ 0.13 D777N/G1207D 1.06 Ϯ 0.36 0.8 Ϯ 0.06 D777N/G1207S 1.12 Ϯ 0.27 2.33 Ϯ 0.14 D777N/S1212L 1.39 Ϯ 0.12 1.36 Ϯ 0.13 D777N/W1225C -b - D777N/R1415G 1.65 Ϯ 0.44 2.02 Ϯ 0.27 a To determine the apparent Km for ATP (mM), the initial rate of LTC4 uptake in vacuolar membrane vesicles was assayed with 50 nM LTC4 and ATP concentrations ranging from 0.035 to 6 mM (see Materials and Methods). Login to comment
220 Eleven of the 13 suppressors isolated are located in the TMDs, not only in the predicted intracytoplasmic loops (A1021T, A1021V, and N1027D) but included in the membrane (V543I, F565L, A1003V, Q1107R, S1212L, and W1225C) or even facing the vacuolar lumen (G1207D and G1207S). Login to comment
229 The results show that a significant fraction of the suppressors,V543I, F565L, A1021T, A1021V, Q1107R, G1207D, S1212L, and R1415G, are deficient for Ycf1p function in cadmium detoxification, pointing to a specific suppression mechanism. Login to comment
234 Phenotypic characterization of the suppressor mutants separated from the primary mutation showed that five of them, namely V543I, F565L, Q1107R, G1207S, and W1225C, exhibit individual different responses to the substrates tested (Fig. 5). Login to comment

[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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Sentences [show]
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). Login to comment
83 We chose to test for GOF effects in these background constructs because F565L was identified as a hit in the aforementioned screen for mutations that suppress defective transport by the Ycf1p Walker B mutant (22). Login to comment
85 As reported previously (22), the F565L mutation partially rescued the cadmium growth phenotype of the NBD1 Walker B construct (D777N). Login to comment
256 Allosteric repair of ATP binding mutants of a long and short MRP The conserved extracellular phenylalanine was chosen for detailed study for 2 reasons: 1) its location at the extracellular ends of the translocations pathways of CFTR and the long and short MRPs permitted us to test for long-range allosteric effects of extracellular perturbations on the apparent ATP occupancies of the cytosolic NBDs of these different transporters (and on the apparent phosphorylation state of the CFTR R domain) and 2) the F565L substitution in Ycf1p (long MRP in yeast) scored as a hit in a previousintragenic screenfor mutations that rescuedpoor growthon cadmium-containing media for yeast expressing the WalkerB mutant, D777N-Ycf1p(22). Login to comment
257 Wereasonedthat the latter result might be related to an allosteric effect of the F565L substitution on ATP occupancy given that the D777N mutation is expected to reduce Mg-ATP binding affinity (15, 22, 36). Login to comment
258 We confirmed this finding and further showed that the same F565L substitution partially rescued the cadmium growth defect exhibited by a second ATP binding mutant, Y1281G-Ycf1p. Login to comment
263 In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment
69 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). Login to comment
82 We chose to test for GOF effects in these background constructs because F565L was identified as a hit in the aforementioned screen for mutations that suppress defective transport by the Ycf1p Walker B mutant (22). Login to comment
84 As reported previously (22), the F565L mutation partially rescued the cadmium growth phenotype of the NBD1 Walker B construct (D777N). Login to comment
255 Allosteric repair of ATP binding mutants of a long and short MRP The conserved extracellular phenylalanine was chosen for detailed study for 2 reasons: 1) its location at the extracellular ends of the translocations pathways of CFTR and the long and short MRPs permitted us to test for long-range allosteric effects of extracellular perturbations on the apparent ATP occupancies of the cytosolic NBDs of these different transporters (and on the apparent phosphorylation state of the CFTR R domain) and 2) the F565L substitution in Ycf1p (long MRP in yeast) scored as a hit in a previousintragenic screenfor mutations that rescuedpoor growthon cadmium-containing media for yeast expressing the WalkerB mutant, D777N-Ycf1p(22). Login to comment
262 In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment