ABCMdb

ABCC1 p.Asp793Leu

Predicted by SNAP2:	A: D (95%), C: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: 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: D,

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[hide] Allosteric interactions between the two non-equiva... J Biol Chem. 2000 Jul 7;275(27):20280-7. Hou Y, Cui L, Riordan JR, Chang X
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
J Biol Chem. 2000 Jul 7;275(27):20280-7., 2000-07-07 [PMID:10781583]

Abstract [show]
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

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33 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L, were generated by using procedures described previously (11). Login to comment

[hide] Nucleotide dissociation from NBD1 promotes solute ... Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61. Yang R, McBride A, Hou YX, Goldberg A, Chang XB
Nucleotide dissociation from NBD1 promotes solute transport by MRP1.
Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61., 2005-03-01 [PMID:15737336]

Abstract [show]
MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.

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7 The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Login to comment
44 Membrane vesicles were prepared from Sf21 cells infected with viral particles expressing pDual without MRP1 cDNA insertion (lane 1), wild-type N-half+wild-type C-half (Wild-type, lanes 2-4), D793E mutated N-half+wild-type C-half (D793E, lanes 5-7), D793L mutated N-half+wild-type C-half (D793L, lanes 8-10), D793N mutated N-half+wild-type C-half (D793N, lanes 11-13) and wild-type N-half+E1455Q mutated C-half (E1455Q, lanes 14-16). Login to comment
50 Since the ratio of N-half, for example, D793E mutated N-half, is similar to that of the C-half co-expressed with D793E mutated N-half, the mean ratios of the protein expressions including N-half and C-half are: 0.993F0.168 (D793E), 0.991F0.073 (D793L), 1.151F0.186 (D793N) and 0.921F0.108 (E1455Q). Login to comment
82 D793L was introduced into the cDNA in the pNUT expression vector already [29]. Login to comment
93 To make constructs expressing D793E, D793L and D793N mutated N-half and wild-type C-half simultaneously, the KpnI- DraIII fragment from pDual/N-half/C-half and the DraIII- XhoI fragments from pNUT/D793E, pNUT/D793L or pNUT/D793N were cloned into the KipI-XhoI fragment from pDual/N-half/C-half, named as pDual/D793E-N-half/ C-half, pDual/D793L-N-half/C-half or pDual/D793N- N-half/C-half. Login to comment
156 Substitution of the Asp residue with a non-acidic amino acid in NBD1 increased the Km and Vmax values for LTC4 in MRP1 mediated transport In order to test whether these Walker B mutations, D793E, D793L and D793N in NBD1 and E1455Q in R. Yang et al. Login to comment
164 Interestingly, the substitution of the putative catalytic base D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increases ATP-dependent LTC4 transport activity (Fig. 3 and Table 1), implying that ATP hydrolysis at NBD1 might not be essential for the transport. Login to comment
169 Since the amounts of MRP1 proteins in membrane vesicles containing wild-type, D793E, D793L, D793N and E1455Q are similar (Fig. 1C), the much lower Vmax value of E1455Q than that of the wild-type (Table 1), although the amount of E1455Q (ratio of 0.921) is slightly less than wild-type, indicates a greatly decreased k2 value, which is perhaps directly associated with the greatly diminished ATPase activity at Fig. 3. Login to comment
174 The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment
175 Table 1 Km and Vmax values (LTC4) of wild-type and mutant MRP1s Sample Km (nM LTC4)a Vmax (pmol LTC4 mgÀ1 minÀ1 )N-half C-half Wild-type Wild-type 59F1 287.5F7.5 D793E Wild-type 110F10 365.0F25.0 D793L Wild-type 100F0 560.0F0.0 D793N Wild-type 105F5 575.0F75.0 Wild-type E1455Q 50F0 37.5F0.5 a The Km values (n=2) and Vmax values (n=2) were derived from Fig. 3. Login to comment
183 / Biochimica et Biophysica Acta 1668 (2005) 248-261 253 the E1455Q mutated NBD2 as shown in Fig. 7M and O; whereas the higher Vmax value (Table 1) of D793L (a ratio of 0.993 indicates that the amount of D793L is slightly less than wild-type) or D793N (ratio of 1.151) indicates a slightly increased k2 value, leading to a higher Km (LTC4) value and a higher rate of ATP-dependent LTC4 transport. Login to comment
185 Combination of D793E, D793L or D793N mutated NBD1 with E1455Q mutated NBD2 does not enhance ATP-dependent LTC4 transport activity The k2 values should be directly associated with the rates of ATP hydrolysis by variant MRP1 mutants. Login to comment
187 Whereas the moderately increased k2 values for D793L and D793N could be interpreted in the following two ways: (1) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, somehow increases the rate of ATP hydrolysis at the mutated NBD1 and enhances ATP-dependent LTC4 transport; (2) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, decreases the affinity for ATP and increases the release rate of the bound nucleotide from the mutated NBD1. Login to comment
189 In order to test these two possibilities, the D793E, D793L or D793N mutated N-half was co-expressed with the E1455Q mutated C-half. Login to comment
191 The results in Fig. 4B show that all the mutants, including wild-type N-half+E1455Q mutated C-half, D793E mutated N-half+E1455Q mutated C-half, D793L mutated N-half+E1455Q mutated C-half and D793N mutated N-half+E1455Q mutated C-half, have similar ATP-dependent LTC4 transport activities. Login to comment
194 D793E, D793L or D793N mutated NBD1 does not enhance the ATP-dependent LTC4 transport activity of E1455Q mutated NBD2. Login to comment
197 The mean ratios of the protein expressions including N-half and C-half are: 1.33F0.11 (E1455Q), 1.49F0.13 (D793E/E1455Q), 0.98F0.05 (D793L/ E1455Q) and 1.88F0.29 (D793N/E1455Q). Login to comment
204 The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment
205 Table 2 Km values (ATP) of wild-type and mutant MRP1s Sample Km (AM ATP)a N-half C-half Wild-type Wild-type 72.2F1.6 D793E Wild-type 106.0F9.7 D793L Wild-type 107.0F7.5 D793N Wild-type 92.0F12.5 Wild-type E1455Q 55.0F0.0 a Km values (for wild-type, D793E, D793L and D793N, n=5; for E1455Q, n=) were derived from corresponding Michaelis-Menten curves shown in Fig. 5. R. Yang et al. Login to comment
210 The Km (ATP) value of E1455Q, the putative catalytic base mutant in NBD2, is slightly less than that of wild-type (Table 2), whereas the Km (ATP) values of D793E, D793L and D793N are slightly higher than that of wild-type (Table 2). Login to comment
220 Fig. 6A, D, G, J and M show the autoradiograms reflecting [a-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the concentration of [a-32 P]-8-N3ATP (Fig. 6B, C, E, F, H, I, K, L, N and O). Login to comment
224 The Kd (ATP) values for D793L and D793N mutated NBD1 co-expressed with wild-type NBD2 are almost three fold higher than that of wild-type NBD1 (Table 3), implying that the mutation of this acidic D793 residue to a non-acidic amino acid decreased k1 (lower rate of binding) and/or increased kÀ1 (higher rate of releasing), i.e. lower affinity. Login to comment
225 The mutation of D793L or D793N does not have a significant effect on the wild-type NBD2 (Table 3). Login to comment
227 In contrast to the counterpart NBD1 mutants D793N and D793L, the substitution of the E1455 with a non-acidic amino acid has an effect on the wild-type NBD1 (Table 3). Login to comment
229 The substitution of the putative catalytic D793 residue with a non-acidic amino acid increases release rate of the bound ATP The higher Kd (ATP) values of D793L and D793N mutated NBD1 were interpreted as that the binding rate was decreased whereas the release rate of the bound ATP was increased. Login to comment
237 Fig. 7A, D, G, J and M show the autoradiograms reflecting [g-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the incubation time (Fig. 7B, C, E, F, H, I, K, L, N and O). Login to comment
247 In the cases of D793E, D793L and D793N, the T1/2 values (for NBD1 and NBD2) are shorter than that of the wild-type and E1455Q (Table 4). Login to comment
264 D793L and D793N mutated NBD1s have higher Kd values than that of wild-type. Login to comment
267 A, D, G, J, and M: Autoradiograms of [a-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment
271 H and I: D793L mutated N-half (H) co-expressed with wild-type C-half (I). Login to comment
274 Table 3 Substitution of D793 with a non-acidic amino acid decreases affinity for ATP Sample Kd of NBD1 (AM ATP)a Kd of NBD2 (AM ATP)N-half C-half Wild-type Wild-type 11.7F2.8 32.7F2.3 D793E Wild-type 7.8F4.1 41.0F8.1 D793L Wild-type 30.5F2.5 32.9F1.9 D793N Wild-type 28.4F4.5 33.7F0.7 Wild-type E1455Q 19.4F3.3 155.8F9.0 a The Kd (AM ATP) values (for wild-type, n=12; D793E, n=9; D793L and E1455Q, n=; D793N, n=8) were derived from Fig. 6. R. Yang et al. Login to comment
287 In contrast, interestingly, the substitution of the putative catalytic residue D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increased the rate of ATP-dependent LTC4 transport ([29] and Fig. 3). Login to comment
288 This radical substitution increased the Kd (ATP) values of the D793L or D793N mutated NBD1 (Fig. 6 and Table 3), where Kd=kÀ1/k1, since ATP hydrolysis on ice is limited. Login to comment
289 The increased Kd of D793N or D793L means increased kÀ1, in other words, increased releasing rate of the bound ATP, and/or decreased k1, in other words, decreased the rate of ATP binding. Login to comment
290 The time required to lose 50% of the bound ATP from the D793L or D793N mutated NBD1 and the co-expressed wild-type NBD2 is much shorter than that of the wild-type (Fig. 7 and Table 4), implying the increased kÀ1 and/or decreased k1 values. Login to comment
301 A, D, G, J, and M: Autoradiograms of [g-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment
307 H and I: D793L mutated N-half (H) co-expressed with wild-type C-half (I). Login to comment
310 Table 4 Release rate of the bound nucleotide at the wild-type and mutated NBDs Sample T1/2 of NBD1 (min)a T1/2 of NBD2 (min)N-half C-half Wild-type Wild-type 5.3F0.3 3.7F2.0 D793E Wild-type 3.0F0.7 3.4F1.0 D793L Wild-type 2.3 2.3 D793N Wild-type 2.5F0.5 2.2F0.7 Wild-type E1455Q 6.1F0.3 25.6F2.4 a The T1/2 value (for wild-type, D793E, D793N and E1455Q, n=3; for D793L, n=1) is the time required to release 50% of the bound nucleotide and was derived from Fig. 7. R. Yang et al. Login to comment

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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262 Interestingly, substitution of the corresponding putative catalytic residue D793 in NBD1 with a non-acidic residue, such as D793L or D793N, increased the rate of ATP-dependent LTC4 transport [139, 144]. Login to comment

[hide] Mutations of the Walker B motif in the first nucle... Arch Biochem Biophys. 2001 Aug 1;392(1):153-61. Cui L, Hou YX, Riordan JR, Chang XB
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
Arch Biochem Biophys. 2001 Aug 1;392(1):153-61., [PMID:11469806]

Abstract [show]
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.

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4 However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. Login to comment
13 Therefore to determine the contribution of NBD1 Walker B aspartate in MRP1 function, we had previously made substitutions at both positions simultaneously, i.e., D792L/D793L (8). Login to comment
17 The results showed that the D792L mutation was responsible for the defective maturation and function while D793L had minimal effect on either. Login to comment
36 The aspartic acid residues at positions of 792 and 793 were mutated either to alanine (Fig. 1B, D792A) or leucine residues (Fig. 1B, D792L and D793L) using the QuikChange Site Directed Mutagenesis kit. Login to comment
42 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). Login to comment
43 The cell lines expressing D792A, D792L, and D793L were generated using the same procedures (9). Login to comment
89 At this level of exposure the mature band is predominantly detected in the wild-type, while none of this band is present in the double mutant, D792L/D793L. Login to comment
90 D793L is indistinguishable from wild-type, whereas D792L is similar to the double mutant indicating that the substitution at this position is primarily responsible for the misprocessing. Login to comment
93 These were more apparent in a longer exposure of the same blot (Fig. 2B), especially in D792L/D793L, where, in addition to the major 170-kDa species, bands of approximately 160, 130, 100, and 30 kDa can be seen. Login to comment
94 Of these four only the 130- and 30-kDa bands are seen in the D792A and D792L lanes. Login to comment
95 Since none of these bands appear in lanes when the protein is fully mature (Fig. 2B, wild-type and D793L), we assume that they are degradation products of the immature forms of the misprocessed mutants. Login to comment
105 To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment
107 By 3 h of chase the wild-type was completely converted to the larger mature form, whereas very little mature D792L and no mature D792L/D793L appeared. Login to comment
124 The following amounts of protein were loaded in each lane: 0.5 ␮g of wild-type MRP1; 8 ␮g of D792A; 18 ␮g of D792L; 0.5 ␮g of D793L; 20 ␮g of D792L/D793L. Login to comment
132 The following amounts were loaded in each lane: 0.5 ␮g of wild-type MRP1; 2 ␮g of D792A; 4 ␮g of D792L; 0.35 ␮g of D793L; 8 ␮g of D792L/D793L. Login to comment
133 The ratio of mature to immature protein in membrane vesicles of D792A and D792L are much higher than in whole cell lysates (Fig. 2A). Login to comment
134 (D) The smaller fragment of D792L/D793L is also core-glycosylated. Login to comment
136 Lanes 1 and 2, 1 ␮g of D793L cell lysates in each lane; Lanes 3 and 4, 4 ␮g of D792A cell lysates in each lane; Lanes 5 and 6, 10 ␮g of D792L/D793L cell lysates in each lane. Login to comment
137 Both the 170-kDa core-glycosylated MRP1 protein from either D793L, D792A, or D792L/D793L and 160-kDa degradation product from D792L/D793L were decreased in size by treatment with endoglycosidase H. pressing either the D792L or D792L/D793L mutants with either lactacystin or ALLN resulted in the total disappearance of the immature forms from nonionic detergent soluble fractions and appearance in insoluble pellets. Login to comment
147 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment
149 In contrast the pattern of disappearance and appearance of bands was entirely different in the case of the D792L mutant (Fig. 5D), which matured poorly and the D792L/D793L mutant that did not mature at all (Fig. 5F). Login to comment
171 Figure 6 shows labeling of the wild-type and the innocuous D793L variant which matures normally. Login to comment
174 Since hydrolysis is believed to drive MRP1 transport it would be expected that the mature D792A protein would not be capable of active transport. Login to comment
175 The data in Fig. 7 confirm this expectation, i.e., there is not significantly more ATP-dependent LTC4 uptake by vesicles containing D792A protein that does mature than by the other variants that do not mature (Fig. 7, D792L and D792L/D793L), nor by the NBD2 mutants (Fig. 7, K1333L and D1454L/E1455L) that do mature but have difficulties to hydrolyze ATP and to trap the hydrolysis product, ADP (8). Login to comment
195 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment
205 (D) D792L, 1.1 ␮g protein in each lane. Login to comment
206 (E) D793L, 0.3 ␮g protein in each lane. Login to comment
207 (F) D792L/D793L, 1.1 ␮g protein in each lane. Login to comment
88 At this level of exposure the mature band is predominantly detected in the wild-type, while none of this band is present in the double mutant, D792L/D793L. Login to comment
92 These were more apparent in a longer exposure of the same blot (Fig. 2B), especially in D792L/D793L, where, in addition to the major 170-kDa species, bands of approximately 160, 130, 100, and 30 kDa can be seen. Login to comment
104 To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment
106 By 3 h of chase the wild-type was completely converted to the larger mature form, whereas very little mature D792L and no mature D792L/D793L appeared. Login to comment
123 The following amounts of protein were loaded in each lane: 0.5 òe;g of wild-type MRP1; 8 òe;g of D792A; 18 òe;g of D792L; 0.5 òe;g of D793L; 20 òe;g of D792L/D793L. Login to comment
131 The following amounts were loaded in each lane: 0.5 òe;g of wild-type MRP1; 2 òe;g of D792A; 4 òe;g of D792L; 0.35 òe;g of D793L; 8 òe;g of D792L/D793L. Login to comment
135 Lanes 1 and 2, 1 òe;g of D793L cell lysates in each lane; Lanes 3 and 4, 4 òe;g of D792A cell lysates in each lane; Lanes 5 and 6, 10 òe;g of D792L/D793L cell lysates in each lane. Login to comment
146 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment
148 In contrast the pattern of disappearance and appearance of bands was entirely different in the case of the D792L mutant (Fig. 5D), which matured poorly and the D792L/D793L mutant that did not mature at all (Fig. 5F). Login to comment
170 Figure 6 shows labeling of the wild-type and the innocuous D793L variant which matures normally. Login to comment
194 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment