Membrane Topology of the Melibiose Permease ofEscherichiacoliStudied bymelB−phoAFusion Analysis

Abstract

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while 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 melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane 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 an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase 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 shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Membrane Topology of the Di- and Tripeptide Transport Protein ofLactococcus lactis

“…Immunodetection of the fusion proteins with high activity revealed, in most cases, proteolytic degradation products the size of native alkaline phosphatase. In general, full length chimeras or degradation products were barely or not detectable with low-activity fusions [as observed by others; e.g., Pourcher et al(1996)]. …”

Excretion and Uptake of Putrescine by the PotE Protein in Escherichia coli

“…The results indicate that both the NH 2 and COOH termini are located in the cytoplasm (see Fig. 5) and that PotE has the same topology as other membrane proteins having 12 transmembrane segments such as lactose permease (39), melibiose permease (40), and the metal-tetracycline/H ϩ antiporter (41).…”

Study of Amide-proton Exchange of Escherichia coliMelibiose Permease by Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy

“…The coupling ions compete for the same binding site and enhance the affinity of the co-transported sugar, with Na ϩ and Li ϩ as better activators than H ϩ (5,6). The currently accepted topological model, consisting of 12 transmembrane domains and N and C termini located in the cytoplasm, is strongly supported by immunological studies (7,8), extensive melBphoA fusion analyses (8,9), and proteolytic mapping (10). Photolabeling (11) and mutagenesis studies combined with fluorescence analysis (12) highlight the importance of the helix IVloop 4 -5 domain for MelB transport function.…”