Membrane topology of the Na+/citrate transporter CitS of Klebsiella pneumoniae by insertion mutagenesis

The sodium ion dependent citrate transporter of Klebsiella pneumoniae (CitS) is a member of the bacterial 2-hydroxycarboxylate transporter family. Membrane topology models of the protein. largely based on reporter molecule fusions to C-terninally truncated CitS molecules, indicate that the protein traverses the membrane 11 times with the NH2-terminus in the cytoplasm and the COOH-terminus in the periplasm. Furthermore, the structure is characterized by unusual long loops in the COOH-terminal half of the protein: one hydrophobic segment between transmembrane segments V and VI in the periplasm and three long loops connecting transmembrane segments VI and VII, VIII and IX and X and XI in the cytoplasm. The 10 kDa biotin acceptor domain and six consecutive His residues (His-tag) were inserted at different positions in the four long loops and the effect on transport activity and protein stability was analyzed. Six out of seven insertion mutants were stably expressed and three of these had retained significant transport activity. The sidedness of the tags in the mutants that tolerated the insertion was determined by proteolysis experiments. The results support the 11 transmembrane segment model that was based upon truncated CitS proteins. (C) 2000 Elsevier Science B.V. All rights reserved.