Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.
Four acidic residues in the N-terminal domain of Na+/proline permease of Escherichia coli (Asp33, Asp34, and Asp55 in putative loop 2, Glu75 in putative transmembrane domain II) were individually replaced with neutral or charged amino acid residues. Replacement of Glu75, the only residue in the permease presumed to be in the middle of a transmembrane domain, Asp33, or Asp34 had little or no influence on the kinetics of Na+-coupled proline transport. In contrast, removal of the carboxylate at position 55 (Asp55 --> Asn or Asp55 --> Cys permease) impaired proline uptake completely while lengthening of the side chain at this position by one methylene group (Asp55 --> Glu permease) allowed transport at a reduced initial rate. Importantly, all permease molecules were present in the membrane at concentrations comparable to the wild-type protein. Kinetic analysis of Na+-coupled proline transport catalyzed by Asp55 --> Glu permease revealed a 5-fold increase of the K(m) for proline and a 30-fold decrease of the V(max) compared to wild-type. Remarkably, replacement of Asp55 by Glu led to a 50-fold decrease of the apparent affinity of the permease for Na+. Furthermore, replacement of Asp55 with Cys or Asn blocked proline-induced Na+ uptake whereas significant Na+ transport was observed with Asp55 --> Glu permease. In addition, transport of proline down its concentration gradient was not detectable with deenergized cells containing Asp55 --> Glu permease at low Na+ concentrations. However, downhill transport activity was observed in the presence of high Na+ concentrations. Replacement of Asp55 with Asn or Cys impaired downhill transport under all conditions tested. The observations demonstrate that a carboxylate at position 55 of proline permease is essential for Na+-coupled proline transport. It is suggested that Asp55 may be involved in binding of the coupling ion.
Quick
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M
Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Germany.
Jung
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H
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011392:Proline / Q000378:metabolism* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D026901:Membrane Transport Proteins; D001224:Aspartic Acid; C033082:proline transporter; D011392:Proline; D012964:Sodium