Transmembrane segment (TMS) VIII of the Na+/citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane

The amino acid sequence of the sodium ion-dependent citrate transporter Cit S of IL pneumoniae contains 12 hydrophobic stretches that could form membra ne-spanning segments. A previous analysis of the membrane topology in Esche richia coli using the PhoA gene fusion technique indicated that only nine o f these hydrophobic segments span the membrane, while three segments, Vb, V III and IX were predicted to have a periplasmic location (Van Geest, IM., a nd Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589), A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes reveal ed that the protein traverses the endoplasmic reticulum membrane 11 times. In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolke ma, J, S, (1999) J. Biol, Chem. 274, 2816-2823), In the present study, using site-directed Cys labeling, the topology of seg ments VIII and IX in the fall-length CitS protein was determined in the E. coli membrane, Engineered cysteine residues in the loop between the two seg ments were accessible to a membrane-impermeable thiol reagent exclusively f rom the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and M are both membrane-spanning. It follows that the folding of CitS in the E. cold and endoplasmic reticulum membrane is the sa me. Cysteine accessibility studies of CitS-PhoA fusion molecules dem; onstr ated that in the E. coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the P hoA fusions do not correctly predict the topology. An engineered cysteine r esidue downstream of TMS VIII moved from a periplasmic to a cytoplasmic loc ation when the fusion protein containing TMSs I-VIII was extended with segm ent IX, Thus, downstream segment M is both essential and sufficient for the insertion of segment VIII of CitS in the E. coli membrane.