Article: article from journal or magazin.
Characterization of the arcD arginine:ornithine exchanger of Pseudomonas aeruginosa. Localization in the cytoplasmic membrane and a topological model.
Journal of Biological Chemistry
The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins.
Alkaline Phosphatase/genetics, Amino Acid Sequence, Amino Acid Transport Systems, Antiporters, Bacterial Proteins/chemistry, Bacterial Proteins/metabolism, Base Sequence, Cloning, Molecular, Colicins/genetics, Cytoplasm/metabolism, DNA, Bacterial, Escherichia coli, Intracellular Membranes/metabolism, Membrane Proteins/chemistry, Membrane Proteins/metabolism, Molecular Sequence Data, Protein Conformation, Pseudomonas aeruginosa/metabolism, Recombinant Fusion Proteins/metabolism
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