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 o f ATP to the translocation of solutes across biological membranes. The so-c alled "Walker motifs" in each of the nucleotide binding domains (NBDs) of t hese proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may int erfere 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 d igestion in vitro than the mature wild-type protein. In vivo, the core-glyc osylated form of that mutant is retained in the endoplasmic reticulum and d egraded by the proteasome. A different substitution of the same residue (D7 92A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but s till resulted in defective nucleotide interaction and organic anion transpo rt, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 func tion. (C) 2001 Academic Press.