Functional Reassembly of the Escherichia coli Maltose Transporter following Purification of a MalF-MalG Subassembly

Abstract

Taking advantage of a chaperone-like function of MalK, a stable complex of MalF-MalG could be solubilized from the Escherichia coli membrane and purified in high yield in the absence of MalK. This MalF-MalG complex was competent for efficient reassembly of a functional MalFGK2 maltose transporter complex both in detergent solution and in proteoliposomes

ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation

“…This result is consistent with a previously reported result that MalK forms a dimer independent of its assembly into the MalFGK 2 complex (23). Although the isolated MalK hydrolyzes ATP slowly (turnover number of 4 per min), the intact MalFGK 2 exhibits greatly enhanced rates, on the order of 10 per sec (24). At a lower protein concentration, 0.1 M, MalK migrates as a monomer in the absence of nucleotides (Fig.…”

MalF is essential for persistence of Mycoplasma gallisepticum in vivo

“…The function of MalF in M. gallisepticum is uncertain, although it appears likely that it is involved in carbohydrate transport into the cell. Its closest homologue in Escherichia coli has been extensively characterized as part of the maltose ABC transporter system (Austermuhle et al, 2004;Caldelari et al, 2008;Cui et al, 2010;Daus et al, 2009;Daus et al, 2007;Daus et al, 2006;Jasco et al, 2009;Mannering et al, 2001;Nikaido, 1994;Sharma et al, 2005). ABC transporters are multi-domain membrane proteins that bind ATP and utilize the energy from its hydrolysis to translocate solutes across cellular membranes (Noll et al, 2008;Pedersen, 2005;Young & Holland, 1999).…”

Nucleotide-free MalK Drives the Transition of the Maltose Transporter to the Inward-facing Conformation

“…For the purification of MalK, E. coli strain BL21 was transformed with pACYC-his MalK, a pACYC184-derivative (New England Biolabs) expressing MalK with an N-terminal 23-amino acid histidine tag extension (13). Transformants were grown at 37°C to A 600 ϳ 0.5 in 12 liters of LB medium containing chloramphenicol (50 g/ml).…”

“…For purification of MalFG, genes of MalF, MalG, and MalK were inserted into the pTrc99A plasmid (Pharmacia). A His 6 tag was placed at the N terminus of MalF and a 23-amino acid histidine tag extension was placed at the N terminus of MalK (yielding pTrc-his FG his K) (13). This plasmid was transformed into E. coli strain BL21.…”

“…2 forms a tight complex in the membrane and, as is often the case for such membrane assemblies, it is difficult to separate the subunits or to express them individually. However, it was reported that MalK modified with a N-terminal 23-amino acid histidine tag extension (termed HT MalK) can dissociate from the complex with urea treatment (13). We therefore expressed HT MalK together with N-terminal His 6 -tagged MalFG (hereafter termed HT MalFG).…”