Membrane Topology of the Sodium Ion-dependent Citrate Carrier of Klebsiella pneumoniae

Abstract

The predicted secondary structure model of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) presents the 12-transmembrane helix motif observed for many secondary transporters. Biochemical evidence presented in this paper is not consistent with this model. N-terminal and C-terminal fusions of CitS with the biotin acceptor domain of the oxaloacetate decarboxylase of K. pneumoniae catalyze citrate transport, showing the correct folding of the CitS part of the fusion proteins in the membrane. Proteolysis experiments with these fusion proteins revealed that the N terminus of CitS is located in the cytoplasm, while the C terminus faces the periplasm. The membrane topology was studied further by constructing a set of 20 different fusions of N-terminal fragments of the citrate transporter with the reporter enzyme alkaline phosphatase (CitS-PhoA fusions). Most fusion points were selected in hydrophilic areas flanking the putative transmembrane-spanning domains in CitS that are predicted from the hydropathy profile of the primary sequence. The alkaline phosphatase activities of cells expressing the CitS-PhoA fusions suggest that the polypeptide traverses the membrane nine times and that the C-terminal half of the protein is characterized by two large hydrophobic periplasmic loops and two large hydrophilic cytoplasmic loops.CitS belongs to the family of the 2-hydroxycarboxylate transporters in which also the citrate carriers, CitPs, of lactic acid bacteria and the malate transporter, MleP, of Lactococcus lactis are found. Since the hydrophobicity profile of CitS is very similar to the hydrophobicity profiles of CitP and MleP, it is most likely that the new structural motif of nine transmembrane segments is shared within this new transporter family.Bacterial secondary solute transporters use the free energy stored in electrochemical gradients of H ϩ and/or Na ϩ to drive the uptake of solutes by coupling the translocation of solute and cation(s) across the membrane. In Klebsiella pneumoniae a unique Na ϩ -dependent citrate carrier (CitS) is induced upon anaerobic growth on citrate (1). The gene coding for the transporter has been cloned and sequenced (2, 3), and the enzyme has been characterized and purified to homogeneity after expression in Escherichia coli. Studies with whole cells of E. coli expressing CitS and with membrane vesicles prepared from these cells have demonstrated that CitS obligatorily couples the translocation of the divalent citrate anion (Hcit 2Ϫ ) to the translocation of two sodium ions and one proton (4, 5). More recent studies with purified CitS (6) reconstituted in proteoliposomes have raised some doubt about the electrogenic nature of the translocation catalyzed by the transporter, and it was concluded that one of the two Na ϩ ions recycle during the translocation of citrate (7).The citS gene encodes a highly hydrophobic protein with a predicted molecular mass of 47,531 Da. The gene is homologous to the genes coding for the citrate carriers of lactic acid bacteria, CitPs (8, ...

Topological Organization of the Hyaluronan Synthase fromStreptococcus pyogenes

“…High-to-intermediate AP activity with low LacZ activity was also observed for fusion proteins at Gly 37 and Asn 60 . These were complemented by fusion proteins at position Thr 7 , Pro 317 , and Lys…”

“…For spHAS-AP fusions, the expression level generally varied inversely with the length of the fusion protein (e.g. fusion constructs at Gly 37 and Asn 60 had 4-fold greater expression levels compared with any other constructs). The only exception to this was for the fusion at position Thr 7 , which had an extremely low expression level for the AP fusion.…”

Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

“…The average and standard deviations of 2-3 independent measurements are reported. 414 in the SSSSC mutant much better in combination than separately, even though the protection was not complete (compare lanes 3,6, and 7 in Fig. 6B).…”

“…Under a constant flow of watersaturated air, and while stirring magnetically, 10 mM potassium ascorbate and 100 M PMS (final concentration) were added, and the proton motive force was allowed to develop for 2 min. Then, [1,[5][6][7][8][9][10][11][12][13][14] C]citrate (114 mCi/mmol Amersham Biosciences) was added to a final concentration of 4.4 M. The uptake was stopped by the addition of 2 ml of ice-cold 0.1 M LiCl, followed by immediate filtration over cellulose nitrate filters (0.45 m, pore size). The filters were washed once with 2 ml of the 0.1 M LiCl solution and assayed for radioactivity.…”

Membrane Topology of the Di- and Tripeptide Transport Protein ofLactococcus lactis

“…The fusion constructs S41, T67, and L343 had PhoA activities that were lower than expected from their blue appearance on XP plates. It should be stressed, however, that the activity was measured in cells that were harvested at the exponential phase of growth whereas the plate assay corresponded to stationary phase cells in which the activity may be higher (van Geest & Lolkema, 1996). All fusion proteins that had a PhoA activity of >75 are indicated by filled circles.…”