Functional consequences of changing proline residues in the phenylalanine-specific permease of Escherichia coli

Abstract

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650-2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [35S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.

FIG. 1

Proposed membrane topology of the PheP permease (11) showing the positions of the 16 proline residues.

FIG. 2

Comparison of levels of wild-type (WT) and proline mutant PheP proteins produced by [³⁵S]methionine-cysteine-labelled JP6488 cells by immunoprecipitation. Representative mutant proteins were pulse-labelled for 1 min before being solubilized with NaOH and immunoprecipitated with an antibody prepared against a synthetic PheP peptide. The radioactivity in gels of immunoprecipitated PheP proteins was quantitated as described in Materials and Methods. Similar values were obtained in two separate experiments.

FIG. 3

Sequence alignment of 13 amino acid transporters of bacteria showing some highly conserved motifs (in boldface) in the region near P341 of PheP.