Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Author(s): Quick M;Quick M; Stölting S; Jung H
  • Source:
    Biochemistry [Biochemistry] 1999 Oct 12; Vol. 38 (41), pp. 13523-9.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370623 Publication Model: Print Cited Medium: Print ISSN: 0006-2960 (Print) Linking ISSN: 00062960 NLM ISO Abbreviation: Biochemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Chemical Society.
    • Subject Terms:
      Amino Acid Transport Systems, Neutral* ; Conserved Sequence*/genetics; Arginine/*chemistry ; Escherichia coli/*enzymology ; Membrane Transport Proteins/*chemistry ; Proline/*metabolism ; Sodium/*metabolism; Amino Acid Sequence ; Amino Acid Substitution/genetics ; Arginine/genetics ; Biological Transport, Active ; Blotting, Western ; Cysteine/chemistry ; Diffusion ; Ethylmaleimide/chemistry ; Kinetics ; Membrane Transport Proteins/genetics ; Membrane Transport Proteins/immunology ; Membrane Transport Proteins/metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed
    • Abstract:
      The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.
    • Accession Number:
      0 (Amino Acid Transport Systems, Neutral)
      0 (Membrane Transport Proteins)
      9040-98-6 (proline transporter)
      94ZLA3W45F (Arginine)
      9DLQ4CIU6V (Proline)
      9NEZ333N27 (Sodium)
      K848JZ4886 (Cysteine)
      O3C74ACM9V (Ethylmaleimide)
    • Publication Date:
      Date Created: 19991016 Date Completed: 19991117 Latest Revision: 20190613
    • Publication Date:
      20231215
    • Accession Number:
      10.1021/bi991256o
    • Accession Number:
      10521259