ABCMdb

ABCC1 p.Ser685Thr

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Publications

PMID: 18088596 [PubMed] Yang R et al: "The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function."

No. Sentence Comment

29 In this report we have mutated S685 to T, A, C, H, D or N and found that only S685T mutation formed complex-glycosylated mature protein, implying that the interaction between the hydroxyl group at 685 in Walker A motif and the carboxyl group at 792 in Walker B motif of NBD1 plays a crucial role for MRP1 protein folding. Login to comment
30 In addition, all these mutants, except S685T, are temperature-sensitive. Login to comment
45 The forward and reverse primers used to introduce these mutations are: S685T/forward, 5'-GTG GGC TGC GGA AAG ACG TCC CTG CTC TCA GCC-3'; S685T/reverse, 5'-GGC TGA GAG CAG GGA CGT CTT TCC GCA GCC CAC-3'; S685A/forward, 5'-GTG GGC TGC GGA AAG GCG TCC CTG CTC TCA GCC-3'; S685A/reverse, 5'-GGC TGA GAG CAG GGA CGC CTT TCC GCA GCC CAC-3'; S685C/forward, 5'-GTG GGC TGC GGA AAG TGC TCC CTG CTC TCA GCC-3'; S685C/reverse, 5'- GGC TGA GAG CAG GGA GCA CTT TCC GCA GCC CAC-3'; S685H/ forward, 5'-GTG GGC TGC GGA AAG CAC TCC CTG CTC TCA GCC-3'; S685H/reverse, 5'-GGC TGA GAG CAG GGA GTG CTT TCC GCA GCC CAC-3'; S685N/forward, 5'- GTG GGC TGC GGA AAG AAC TCC CTG CTC TCA GCC-3'; S685N/reverse, 5'-GGC TGA GAG CAG GGA GTT CTT TCC GCA GCC CAC-3'; S685D/forward, 5'-GTG GGC TGC GGA AAG GAT TCC CTG CTC TCA GCC-3'; S685D/reverse, 5'-GGC TGA GAG CAG GGA ATC CTT TCC GCA GCC CAC-3'. Login to comment
98 Substitution of the S685 with an amino acid that prevents formation of the hydrogen-bond with D792, such as S685A, Table 1 Km (Mg·ATP) and Vmax (LTC4) Values of wild-type and mutant MRP1s Vmax (pmol/mg/min)* Km (μM)* MRP1 164.0±7.0 59.0±2.2 S685T 330.7±8.8 143.0±8.2 S685D 65.3±1.2 249.3±6.3 D792S 79.3±2.1 245.3±8.2 S685D/D792S 99.0±2.9 151.3±6.8 *Km (Mg·ATP) and Vmax (LTC4) values for wild-type, S685T, S685D, D792S and S685D/D792S (n=3) were derived from the corresponding Michaelis-Menten curves shown in Fig. 6. Login to comment
99 The P value for comparison of Km (Mg·ATP) of wild-type versus S685T is 0.0001 and, therefore, the P values for other mutants must be at most 0.0001. Login to comment
110 In order to test whether the hydroxyl group in S685 plays such an important role for the protein folding, this serine residue was mutated to threonine (S685T in Fig. 2A). Login to comment
111 Interestingly, S685T mutant produced similar amount of complex-glycosylated mature protein as wild-type (Fig. 2B), implying that the hydroxyl group in threonine plays a similar role as the one in serine residue. Login to comment
121 However, the amounts of MRP1 mutant proteins accumulated in BHK cells at 27 °C is still much less than that of wild-type or S685T (Fig. 3A), probably due to the mutants are not as stable as wild-type, even at 27 °C. Login to comment
122 This hypothesis is confirmed by the fact that the two degradation products, ~75 kDa and ~35 kDa, were clearly detected by mAb against NBD2 in the mutants of S685H, S685N, S685C or S685A grown at 27 °C, but not in wild-type or S685T (Fig. 3A). Login to comment
155 Although the amounts of MRP1 mutants in the membrane vesicles are much less than that of wild-type or S685T (Fig. 5A), majorities of these mutants in membrane vesicles prepared from the cells grown at 27 °C are complex-glycosylated mature MRP1s (Fig. 5A). Login to comment
157 For un-known reasons, the ATP-dependent LTC4 transport activity catalyzed by S685T is significantly Fig. 4. Login to comment
198 As shown in Fig. 6 and Table 1, the Vmax (LTC4) value of S685T is significantly higher than that of wild-type, consistent with the results derived from a solution containing 4 mM ATP and 10 mM MgCl2 (Fig. 5B). Login to comment
199 Although the exact mechanism of why S685T is more active than the wild-type is not clear, the higher Km (Mg·ATP) value of S685T (Table 1) might be a factor. Login to comment
200 However, the Km (Mg·ATP) values of S685D, D792S and S685D/D792S are even higher than that of S685T (Table 1), whereas the Vmax (LTC4) values of these mutants are much lower than that of wild-type. Login to comment
213 S685A or S685T mutation was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells [24]. Login to comment
216 wild-type, S685A-, S685T-, S685D-, D792S- and S685D/ D792S-mutated MRP1s were used to do photo-affinity labeling at 37 °C with either [α-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP in the presence of vanadate. Login to comment
223 In contrast, the labeling of S685T with [γ-32 P]-8-N3ATP is significantly higher than the corresponding labeling on wild-type MRP1 (Fig. 7A). Login to comment
224 In addition, the labeling of S685T with [α-32 P]-8-N3ATP is also higher than the corresponding labeling on wild-type MRP1 (Fig. 7A). Login to comment
225 The ratio of [γ-32 P]-8-N3ATP labeling, after subtracting the labeling without UV-irradiation, versus [α-32 P]-8-N3ATP labeling on wild-type MRP1 is approximately 0.3, whereas this ratio for S685T is approximately 0.6, implying that ATP bound to the S685T-mutated MRP1 may not be efficiently hydrolyzed. Login to comment
226 However, the ATP-dependent LTC4 transport activity of S685T-mutated MRP1 is much higher than that of wild-type (Figs. 5 and 6). Login to comment
227 In order to solve this puzzle, S685T was introduced into pDual/N-half/ C-half expression vector [24,25] and expressed in Sf21 cells. Login to comment
232 In contrast, the [γ-32 P]-8-N3ATP or [α-32 P]-8-N3ATP labeling of S685T-mutated-NBD1-containing N-half is significantly higher than the corresponding labeling on the wild-type N-half (Fig. 7B), presumably the S685T mutation increased affinity for 8-N3ATP at the mutated NBD1. Login to comment
233 In addition, the [γ-32 P]-8-N3ATP labeling of S685T-mutated- NBD1-containing N-half is much higher than that of the labeling on the un-mutated NBD2-containing C-half (Fig. 7B) and the [α-32 P]-8-N3ATP labeling of the un-mutated NBD2-containing C-half of S685T-mutated MRP1 is much higher than the [γ-32 P]-8-N3ATP labeling of the same fragment (Fig. 7B), indicating that ATP bound to this NBD2 is efficiently hydrolyzed. Login to comment
250 In contrast to other mutations, substitution of the Walker A serine residue with a threonine (S685T) that does not change the position of the hydroxyl group, except introducing an extra methyl group, results in complex-glycosylated mature MRP1 protein (Figs. 2B and 3). Login to comment
264 In addition, substitution of the Walker A serine residue with a threonine (S685T) without changing the position of the hydroxyl group exerted higher affinity for 8-azido-ATP (Fig. 7) at the mutated NBD1 and significantly increased ATP-dependent LTC4 transport activity (Table 1 and Figs. 5 and 6), emphasizing the importance of the hydroxyl group located at this position. Login to comment