Functional analysis of an inosine-guanosine transporter from Leishmania donovani: The role of conserved residues, aspartate 389 and arginine 393

Shirin Arastu-Kapur, Ethan Ford, Buddy Ullman, Nicola Carter

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp 389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into δldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp 389 and ARg393 interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/ R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/R393D localized properly to the plasma membrane, while neither D389R/ R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp389 is critical for optimal transporter function and that a positively charged or polar residue at Arg393 is essential for proper expression of LdNT2 at the plasma membrane.

Original languageEnglish (US)
Pages (from-to)33327-33333
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number35
DOIs
StatePublished - Aug 29 2003

Fingerprint

Leishmania donovani
Inosine
Functional analysis
Guanosine
Nucleoside Transport Proteins
Aspartic Acid
Arginine
Parasites
Cell membranes
Green Fluorescent Proteins
Cell Membrane
Membranes
Flagella
Mutant Proteins
Transfection
Phenotype
Mutation
arginine aspartate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional analysis of an inosine-guanosine transporter from Leishmania donovani : The role of conserved residues, aspartate 389 and arginine 393. / Arastu-Kapur, Shirin; Ford, Ethan; Ullman, Buddy; Carter, Nicola.

In: Journal of Biological Chemistry, Vol. 278, No. 35, 29.08.2003, p. 33327-33333.

Research output: Contribution to journalArticle

Arastu-Kapur, Shirin ; Ford, Ethan ; Ullman, Buddy ; Carter, Nicola. / Functional analysis of an inosine-guanosine transporter from Leishmania donovani : The role of conserved residues, aspartate 389 and arginine 393. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 35. pp. 33327-33333.
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