Abstract

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V-max of 1.25 mumol P-i/min/mg and a K-m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/ mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQI40K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes. (C) 2002 Elsevier Science B.V. All rights reserved.

Keywords

ABC transporter; maltose transport; binding protein-dependent; proteoliposomes; assembly; Salmonella typhimurium

Published in

BBA - Biomembranes
2002, Volume: 1565, number: 1, article number: PII S0005-2736(02)00506-0

UKÄ Subject classification

Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1016/s0005-2736(02)00506-0

Permanent link to this page (URI)

https://res.slu.se/id/publ/102693