A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport.
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.
Cosgriff
A J
AJ
Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, 3052, Australia.
Brasier
G
G
Pi
J
J
Dogovski
C
C
Sarsero
J P
JP
Pittard
A J
AJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000145:classification / Q000378:metabolism*; D011995:Recombination, Genetic; D014364:Tryptophan / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; C063201:AroP protein, E coli; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C069320:phenylalanine-specific permease, E coli; D014443:Tyrosine; D014364:Tryptophan; D000469:Alkaline Phosphatase