Characterization of the arcD arginine:ornithine exchanger of Pseudomonas aeruginosa. Localization in the cytoplasmic membrane and a topological model.
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.
Bourdineaud
J P
JP
Department of Microbiology, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Heierli
D
D
Gamper
M
M
Verhoogt
H J
HJ
Driessen
A J
AJ
Konings
W N
WN
Lazdunski
C
C
Haas
D
D
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems; D017920:Antiporters; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003087:Colicins / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D004269:DNA, Bacterial; D004926:Escherichia coli; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D026905:Amino Acid Transport Systems; D017920:Antiporters; C063930:ArcD protein, Bacteria; D001426:Bacterial Proteins; D003087:Colicins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase