TOPOLOGICAL STUDY OF VIBRIO-ALGINOLYTICUS NHAB NA+ H+ ANTIPORTER USING GENE FUSIONS IN ESCHERICHIA-COLI-CELLS/

NhaB, an Na+/H+ antiporter, of Vibrio alginolyticus is a 528-amino-aci d protein. Hydropathy profile-based computer analysis predicted that t he NhaB might contain up to 13 membrane-spanning domains. To examine t his hypothesis, we applied the phoA fusion method to the cloned nhaB g ene. Eighteen plasmid-borne nhaB-phoA fusion genes were constructed in Escherichia coli cells and the alkaline phosphatase activity and expr ession level of the fusion proteins analyzed. These results and the re sults obtained with additional constructs indicated that V. alginolyti cus NhaB has a unique topology consisting of nine transmembrane segmen ts with the N-terminus in the cytoplasm and the C-terminus in the peri plasm. (C) 1998 Elsevier Science B.V.