Isolation of an ABC transporter, LolCDE, possessing tightly bound lipoproteins

　　Escherichia coli lipoproteins are anchored to the periplasmic side of either the inner or outer membrane through lipid moiety attached to the amino-terminal cystein. LolCDE, an ABC transporter, releases the outer membrane directed lipoproteins from the inner membrane in an ATP-dependent manner, leading to the formation of a complex between lipoprotein and periplasmic chaperone LolA. The LolCDE complex comprises two copies of ATPase subunits, LolD, and one copy each of integral membrane subunits, LolC and LolE. Both LolC and LolE span the membrane four times and possess a large periplasmic domain. We found that LolCDE can be purified with lipoproteins tightly bound to it. As far as we know, this is the first example of an ABC transporter that is purified with its substrate. Our data suggest that this liganded-LolCDE complex is an intermediate of lipoprotein release reaction. We also analyzed the LolD region that is important for the functional interaction with LolCE.
　　Membranes prepared from LolCDHisE overproducing cells were solubilized with a detergent, and subjected to a TALON resin. Unliganded-LolCDHE was purified in the presence of ATP whereas LolCDHE obtained in the absence of ATP contained lipoproteins, suggesting that LolCDHE can bind ligands prior to ATP. Liganded-LolCDHE reconstituted into proteoliposomes released associated lipoproteins upon the addition of LolA/ATP, indicating that the liganded-LolCDHE represents an intermediate of the release reaction.
　　ABC transporters can be classified based on the sequence differences in the helical domain located between the Walker motifs A and B. It is the helical domain, rather than the Walker and Signature motifs, that determines the functional and structural characteristics of each ABC transporter family. "LolD motif" region comprising 32 amino acid residues is highly conserved among LolD homologs and speculated to be important for the interaction with membrane subunits. We constructed 30 species of dominant-negative mutants that have an alteration in the LolD motif region. The LolCDE complex containing LolD mutants, Y93H H96P and H97N, had very low ATPase activity. On the other hand, these mutants exhibited substantial activity when they were not in the complex. We also isolated mutants that can suppress the LolD motif mutations. Many suppressors were found to possess mutations in the first cytoplasmic loop and the second transmembrane segment of LolE, suggesting that these regions of LolE interact with LolD motif region.