J. Bacteriol., May 1996, 2650-2655, Vol 178, No. 9
Copyright © 1996, American Society for Microbiology

Topology of the phenylalanine-specific permease of Escherichia coli

J Pi and AJ Pittard
Department of Microbiology, The University of Melbourne, Parkville, Victoria, Australia.

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site- directed mutagenesis of some of the residues involved.

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