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Journal of Bacteriology, April 2000, p. 2207-2217, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Angela J. Cosgriff,dagger Geoff Brasier, Jing Pi, Con Dogovski, Joseph P. Sarsero,Dagger and A. J. Pittard*

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, 3052, Australia

Received 28 December 1999/Accepted 1 February 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, 3052, Australia. Phone: 61 3 9344 5679. Fax: 61 3 9347 1540. E-mail: aj.pittard{at}microbiology.unimelb.edu.au.

dagger Present address: Drug Development, AMRAD Operations, Richmond, Victoria, 3121, Australia.

Dagger Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020.

Journal of Bacteriology, April 2000, p. 2207-2217, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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