Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium.
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 micromol 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 MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.
Landmesser
Heidi
H
Humboldt Universität zu Berlin, Institut für Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115, Berlin, Germany.
Stein
Anke
A
Blüschke
Bettina
B
Brinkmann
Melanie
M
Hunke
Sabine
S
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008320:Maltose / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011510:Proteolipids; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014508:Urea; D014638:Vanadates / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C468279:MalFGK(2) protein, Salmonella typhimurium; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D014638:Vanadates; D008320:Maltose; D014508:Urea; D000251:Adenosine Triphosphatases