ABCMdb

PMID: 11466279

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., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 55 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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. Login to comment 65 ABCC1 p.Phe565Leu 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 ABCC1 p.Phe565Leu 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 101 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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. Login to comment 108 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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). Login to comment 111 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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. Login to comment 120 ABCC1 p.Gly1207Ser ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp Certain mutations were detected more than once; these included A1021V (four times), A1021T (three), G1207D (two), G1207S (two), and W1225C (seven). Login to comment 130 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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. Login to comment 132 ABCC1 p.Gly1207Ser ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Ala1021Val ABCC1 p.Ala1021Val ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp ABCC1 p.Gly1207Asp 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 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Gly1207Asp 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 ABCC1 p.Gly1207Ser ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp ABCC1 p.Gly1207Asp 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 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Gly1207Asp 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 186 ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 187 ABCC1 p.Phe565Leu 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 188 ABCC1 p.Gly1207Ser Third, mutant G1207S displayed a Cd2ϩ resistance similar to that of the wild-type cells but an enhanced tolerance to diamide. Login to comment 190 ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 191 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu 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 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 204 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp We performed a revertant analysis of five mutations located in the NBDs of Ycf1p, namely G663V, G756D, D777N, G1306E, and G1311R. Login to comment 207 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp We were unable to identify any intragenic suppressors of the other four alleles, G663V, G756D, G1306E, and G1311R. Login to comment 212 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp Moreover, the NBD1 mutations G663V and G756D were not suppressed when combined with some of the suppressors isolated for D777N (see below). Login to comment 220 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 ABCC1 p.Phe565Leu ABCC1 p.Ala1021Thr ABCC1 p.Ala1021Val ABCC1 p.Gly1207Asp 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 230 ABCC1 p.Gly663Val ABCC1 p.Gly756Asp 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). Login to comment 232 ABCC1 p.Gly1207Ser In contrast, mutations G1207S and W1225C produce an enhanced Ycf1p function even in the absence of D777N mutation, indicating a different mechanism of suppression that involves a change in Ycf1p substrate specificity. Login to comment 234 ABCC1 p.Gly1207Ser ABCC1 p.Phe565Leu 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