Insertion of a bacterial secondary transport protein in the endoplasmic reticulum membrane

The sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) co ntains 12 hydrophobic potential transmembrane domains. Surprisingly, an alk aline phosphatase fusion study in Escherichia coil has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589), To provide independent data on the topology and mode of memb rane insertion of CitS, we have investigated its insertion into the endopla smic reticulum (ER) membrane. By using in vitro translation of model protei ns in the presence of dog pancreas microsomes, each of the putative transme mbrane segments of CitS was assayed for its potency to insert into the ER m embrane, both as an isolated segment as well as in the context of COOH-term inal truncation mutants. All 12 segments were able to insert into the membr ane as N-cyt- C-lum signal anchor sequences, In a series of COOH-terminal t runcation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segm ents VIII and M, which, according to the alkaline phosphatase fusion study, are in the periplasm off, coil, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segme nts and also demonstrate that the sequence requirements for signal anchor a nd stop transfer function are different.