Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli

Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed.The GabP and PheP proteins are members of the amino acid transport (AAT) family within the superfamily of amino acidpolyamine-organocation transporters (15). Approximately 30% of the residues are identical in these two proteins. Both GabP and PheP are polytopic integral membrane proteins containing 12 membrane-spanning regions, as determined by hydropathy plots and studies of fusion proteins (5, 7). GabP permease is primarily a transporter for ␥-aminobutyric acid, and PheP is primarily a transporter for the aromatic amino acids phenylalanine and tyrosine (4,8).Hu and King (2) have identified a sensitive polar surface (SPS) extending from the latter half of transmembrane span VIII into the following cytoplasmic loop of GabP, which is part of a consensus amphipathic region (CAR) and is postulated to play a major role in substrate translocation (2). In the case of PheP, the cytoplasmic loop between transmembrane spans VIII and IX has been shown to be very sensitive to single amino acid insertions and to contain two highly conserved amino acid residues, R317 and P341, each of which is critical for protein function (6, 9). Since Hu and King have suggested that the role proposed for the CAR in GabP may also apply to all other members of the AAT family, we carried out a similar analysis of the putative SPS in PheP to test the generality of the results obtained with GabP (2). This analysis revealed three residues in this region of PheP that are critical for protein function and a fourth, in an earlier cytoplasmic loop, with which one of these three residues may interact. Starting with primary mutants with mutations at one of these three positions and also with mutants with mutations at the previously studied residue P341, we selected and analyzed a collecti...

mentioning

confidence: 99%

Study of Second-Site Suppression in the pheP Gene for the Phenylalanine Transporter of Escherichia coli

Chow

Pittard

2002

“…AroP, together with the closely related phenylalanine-specific permease PheP, belongs to a superfamily of permeases involved in the transport of amino acids in bacteria and yeast (32,33).…”

mentioning

confidence: 99%

A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli

Cosgriff

Pittard

1997

Self Cite

The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan. A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease. The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase. Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions. In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5. These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity. It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.The general aromatic amino acid permease, AroP, of Escherichia coli is an integral cytoplasmic membrane protein involved in the active transport of phenylalanine, tyrosine, and tryptophan (8,12,46). AroP, together with the closely related phenylalanine-specific permease PheP, belongs to a superfamily of permeases involved in the transport of amino acids in bacteria and yeast (32,33).A possible model for the secondary structure of the AroP permease in the cytoplasmic membrane has been proposed based on the hydrophobicity profile and distribution of charged amino acid residues. In this model the hydrophobic nonpolar residues are arranged in 12 membrane-spanning regions of approximately 21 amino acids connected by hydrophilic loops of various lengths (14). This proposed topological model is very similar to that reported for the PheP permease (31), which has 61% sequence identity with the AroP permease ( Fig. 1). Despite the high degree of sequence identity and similarity in hydrophobicity profiles, AroP and PheP differ in their range of substrate specificities and affinities. While PheP shows a specific activity for transporting phenylalanine, AroP is equally capable of transporting each of the three aromatic amino acids.In order to validate the proposed topological model of AroP and to determine whether there are major differences between the membrane topologies of AroP and PheP, alkaline phosphatase was used as a reporter enzyme to analyze the membrane topology of the AroP permease. Alkaline phosphatase is enzymatically active only when translocated across the cytoplasmic membrane into the periplasm (28), where its intrachain disulfide bonds can form (15). In the absence of its leader peptide, this translocation can occur only if alkaline phosphatase is fused to a signal sequence or to a periplasmic domain of a membrane protein, with such fusions exhibiting high-level enzymatic activity. However, when t...

“…At the position corresponding to 118 in PheP, other bacterial members of the family contain either glutamate or aspartate. Converting glutamate 118 of the PheP protein to glycine, valine, leucine, tryptophan, or asparagine has been shown to completely destroy transport activity, whereas changing it to aspartate reduces transport activity to 36% of wild-type level (24). These results indicate the importance of glutamate 118, which is presumed to have a critical role either in proton translocation or in substrate binding or both.…”

Section: Discussionmentioning

confidence: 58%

“…Glutamate 118, whose possible function is discussed below, is a highly conserved residue whose replacement by residues other than aspartate leads to loss of function (24).…”

Section: Resultsmentioning

confidence: 99%

“…When six of the aromatic residues were changed individually to leucine, mutants involving phenylalanine 98 or tyrosine 107 showed no change in phenylalanine transport, but changes to either phenylalanine 101, tryptophan 105, tryptophan 108, or phenylalanine 111 resulted in a loss of between 40 and 60% of transport activity (24). In considering whether or not these amino acids had an important role to play in a putative channel for the aromatic amino acids, we had concluded that this loss of activity was not a sufficient indication.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Study of AroP-PheP Chimeric Proteins and Identification of a Residue Involved in Tryptophan Transport

Cosgriff

Brasier

et al. 2000

In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.