Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
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.
Cui
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Mayo Clinic Scottsdale, S. C. Johnson Medical Research Center, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.
Hou
Y X
YX
Riordan
J R
JR
Chang
X B
XB
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D009711:Nucleotides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
D018528:ATP-Binding Cassette Transporters; D003546:Cysteine Endopeptidases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009097:Multienzyme Complexes; D009711:Nucleotides; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins; D014357:Trypsin