FUNCTIONAL CONSEQUENCES OF CHANGING PROLINE RESIDUES IN THE PHENYLALANINE-SPECIFIC PERMEASE OF ESCHERICHIA-COLI

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 p hosphatase has previously been proposed in which PheP has 12 transmemb rane segments with both N and C termini located in the cytoplasm (J, P i 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 a lanine at only three positions, P54, P341, and P442, resulted in the l oss of 50% or more activity. Substitutions at P341 had the most dramat ic effects, None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into th e membrane, as indicated by [S-35]methionine labelling and immunopreci pitation using anti-PheP serum. A possible role for each of these thre e prolines is discussed, Inserting a single alanine residue at differe nt 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.