ABCMdb

ABCC1 p.Ala1021Val

Predicted by SNAP2:	C: N (72%), D: D (71%), E: D (75%), F: D (66%), G: N (82%), H: D (75%), I: N (53%), K: D (80%), L: N (72%), M: N (53%), N: D (63%), P: D (80%), Q: D (71%), R: D (80%), S: N (82%), T: N (82%), V: N (82%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	C: D, D: D, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D,

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Publications
[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
120 Certain mutations were detected more than once; these included A1021V (four times), A1021T (three), G1207D (two), G1207S (two), and W1225C (seven). 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
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
186 First, mutants A1021T, A1021V, G1207D, S1212L, and R1415G were more sensitive than the wild-type strain to inhibition by both Cd2ϩ and diamide, with 35 to 80% decreases in the MICs of these substrates when compared to those for the wild type. Login to comment
190 Thus, the resistance phenotype analysis of the second-site mutants showed a group of nonfunctional mutants for both substrates, A1021T, A1021V, G1207D, S1212L, and R1415G, indicating that Ala1021, Glu1207, Ser1212, and Arg1415 are functionally important residues in Ycf1p. 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