MEMBRANE TOPOLOGY OF THE MELIBIOSE PERMEASE OF ESCHERICHIA-COLI STUDIED BY MELB-PHOA FUSION ANALYSIS

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assa yed for alkaline phosphatase activity. In general agreement with a pre viously suggested secondary structure model of melibiose permease [Bot field, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T. H. (1999) J. Bio l. Chem. 267, 1818], clusters of fusions exhibiting low and high phosp hatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in th e periplasmic half of transmembrane domains or in periplasmic loops, w hile fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that mel ibiose permease contains 12 transmembrane domains that traverse the me mbrane in zigzag fashion connected by hydrophilic loops that are expos ed alternatively on the periplasmic or cytoplasmic surfaces of the mem brane with the N and C termini on the cytoplasmic face of the membrane . Moreover, on the basis of the finding that the cytoplasmic half of a n out-going segment is sufficient for alkaline phosphatase export to t he periplasm while the periplasmic half of an in-going segment prevent s it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S,A. 8 7, 4837], the activity profile of the melibiose permease-alkaline phos phatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shif ted from a hydrophilic to a hydrophobic domain in this region of the t ransporter.