ABCMdb

PMID: 11469806

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 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 5 ABCC1 p.Asp792Leu We have determined now that this latter effect is entirely due to the D792L substitution. Login to comment 8 ABCC1 p.Asp792Ala 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. Login to comment 13 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 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 18 ABCC1 p.Asp792Ala A different substitution of the important residue (D792A) only partially impaired maturation yet still 1 To whom correspondence and reprint requests should be addressed. Login to comment 36 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp1454Leu ABCC1 p.Asp792Leu ABCC1 p.Lys684Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). Login to comment 43 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala The cell lines expressing D792A, D792L, and D793L were generated using the same procedures (9). Login to comment 88 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 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 89 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 91 ABCC1 p.Asp792Ala However, when this residue was replaced by alanine (D792A) instead of leucine a detectable mature band was also present. Login to comment 92 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 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 93 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala Of these four only the 130- and 30-kDa bands are seen in the D792A and D792L lanes. Login to comment 95 ABCC1 p.Asp793Leu 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 98 ABCC1 p.Asp792Ala It is also apparent that the mature form of MRP1 is enriched in the membrane fraction compared with the whole cell lysate; this is especially obvious with D792A, where there appears to be about equal amounts of the immature and mature bands in the membrane fraction (Fig. 2C). Login to comment 99 ABCC1 p.Asp792Ala It is also apparent that the mature form of MRP1 is enriched in the membrane fraction compared with the whole cell lysate; this is especially obvious with D792A, where there appears to be about equal amounts of the immature and mature bands in the membrane fraction (Fig. 2C). Login to comment 104 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment 105 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment 106 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu 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 107 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu 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 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 124 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 131 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 132 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu (D) The smaller fragment of D792L/D793L is also core-glycosylated. Login to comment 135 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 136 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala 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 146 ABCC1 p.Asp793Leu ABCC1 p.Asp1454Leu ABCC1 p.Lys684Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu 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 147 ABCC1 p.Asp793Leu ABCC1 p.Asp1454Leu ABCC1 p.Lys684Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu 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 149 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu 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 152 ABCC1 p.Asp792Leu This smaller product of digestion of the immature D792L polypeptide appeared at a lower trypsin to membrane protein ratio (Fig. 5D, lane 6, 1:128). Login to comment 153 ABCC1 p.Asp792Leu This smaller product of digestion of the immature D792L polypeptide appeared at a lower trypsin to membrane protein ratio (Fig. 5D, lane 6, 1:128). Login to comment 154 ABCC1 p.Asp792Ala This comparison is most obvious with the D792A mutant where the membranes subjected to trypsin digestion contain approximately equal amounts of immature and mature species (Fig. 5C). Login to comment 155 ABCC1 p.Asp792Ala This comparison is most obvious with the D792A mutant where the membranes subjected to trypsin digestion contain approximately equal amounts of immature and mature species (Fig. 5C). Login to comment 158 ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu The D792L mutant is nearly and the D792L/D792L mutant is completely unable to undergo this conformation change. Login to comment 159 ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu The D792L mutant is nearly and the D792L/D792L mutant is completely unable to undergo this conformation change. Login to comment 167 ABCC1 p.Asp792Leu It was impossible to tell if the dysfunction of the D792L mutation was secondary to its inability to mature conformationally. Login to comment 168 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala It was impossible to tell if the dysfunction of the D792L mutation was secondary to its inability to mature conformationally. Login to comment 169 ABCC1 p.Asp792Ala However, since approximately half of the D792A protein in the membrane was mature it was possible to assay its functional capability. Login to comment 170 ABCC1 p.Asp793Leu Figure 6 shows labeling of the wild-type and the innocuous D793L variant which matures normally. Login to comment 171 ABCC1 p.Asp793Leu ABCC1 p.Asp792Ala Figure 6 shows labeling of the wild-type and the innocuous D793L variant which matures normally. Login to comment 172 ABCC1 p.Asp792Ala However, there was greatly reduced labeling of the variants in which D792 was substituted including D792A where there is considerable mature protein. Login to comment 173 ABCC1 p.Asp792Ala 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 174 ABCC1 p.Asp793Leu ABCC1 p.Asp1454Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu ABCC1 p.Asp792Ala ABCC1 p.Asp792Ala 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 ABCC1 p.Asp793Leu ABCC1 p.Asp1454Leu ABCC1 p.Asp792Leu ABCC1 p.Asp792Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu ABCC1 p.Asp792Ala 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 180 ABCC1 p.Asp792Leu The D792L mutant had minimal ability to trap nucleotide and transport an organic anion but it was also unable to mature during biosynthesis. Login to comment 181 ABCC1 p.Asp792Leu The D792L mutant had minimal ability to trap nucleotide and transport an organic anion but it was also unable to mature during biosynthesis. Login to comment 183 ABCC1 p.Asp792Ala However, when a different substitution, D792A, was made considerable maturation occurred although still less than wild-type (Figs. 2B and 2C). Login to comment 184 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala However, when a different substitution, D792A, was made considerable maturation occurred although still less than wild-type (Figs. 2B and 2C). Login to comment 185 ABCC1 p.Asp792Leu Nevertheless, this mutant was not capable of greater transport activity than the completely misprocessed D792L (Fig. 7). Login to comment 194 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment 195 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment 202 ABCC1 p.Lys684Leu (B) K684L, 0.6 òe;g protein in each lane. Login to comment 203 ABCC1 p.Lys684Leu ABCC1 p.Asp792Ala (B) K684L, 0.6 ␮g protein in each lane. Login to comment 204 ABCC1 p.Asp792Leu ABCC1 p.Asp792Ala (C) D792A, 0.73 ␮g protein in each lane. Login to comment 205 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu (D) D792L, 1.1 ␮g protein in each lane. Login to comment 206 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu (E) D793L, 0.3 ␮g protein in each lane. Login to comment 207 ABCC1 p.Asp793Leu ABCC1 p.Asp792Leu ABCC1 p.Lys1333Leu (F) D792L/D793L, 1.1 ␮g protein in each lane. Login to comment 208 ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu ABCC1 p.Lys1333Leu (G) K1333L, 0.3 ␮g protein in each lane. Login to comment 209 ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu (H) D1454L/E1455L, 0.3 ␮g protein in each lane. Login to comment 215 ABCC1 p.Asp792Leu This appears to occur with the D792L variant of MRP1 (Fig. 4). Login to comment 216 ABCC1 p.Asp792Leu This appears to occur with the D792L variant of MRP1 (Fig. 4). Login to comment