MEMBRANE TOPOLOGY OF THE SODIUM ION-DEPENDENT CITRATE CARRIER OF KLEBSIELLA-PNEUMONIAE - EVIDENCE FOR A NEW STRUCTURAL CLASS OF SECONDARY TRANSPORTERS

The predicted secondary structure model of the sodium ion-dependent ci trate carrier of Klebsiella pneumoniae (CitS) presents the la-transmem brane helix motif observed for many secondary transporters, Biochemica l evidence presented in this paper is not consistent with this model. N-terminal and C-terminal fusions of CitS with the biotin acceptor dom ain of the oxaloacetate decarboxylase of K. pneumoniae catalyze citrat e transport, showing the correct folding of the CitS part of the fusio n proteins in the membrane. Proteolysis experiments with these fusion proteins revealed that the N terminus of CitS is located in the cytopl asm, while the C terminus faces the periplasm, The membrane topology w as 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 sel ected in hydrophilic areas flanking the putative transmembrane-spannin g domains in CitS that are predicted from the hydropathy profile of th e primary sequence, The alkaline phosphatase activities of cells expre ssing the CitS-PhoA fusions suggest that the polypeptide traverses the membrane nine times and that the C-terminal half of the protein is ch aracterized by two large hydrophobic periplasmic loops and two large h ydrophilic cytoplasmic loops. CitS belongs to the family of the 2-hydr oxycarboxylate transporters in which also the citrate carriers, CitPs, of lactic acid bacteria and the malate transporter, MleP, of Lactococ cus lactis are found. Since the hydrophobicity profile of CitS is very similar to the hydrophobicity profiles of CitP and MleP, it is most l ikely that the new structural motif of nine transmembrane segments is shared within this new transporter family.