Functional analysis of an inosine-guanosine transporter from Leishmania donovani. The role of conserved residues, aspartate 389 and arginine 393.
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 Asp389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Delta ldnt2 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 Asp389 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.
Arastu-Kapur
Shirin
S
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Ford
Ethan
E
Ullman
Buddy
B
Carter
Nicola S
NS
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D005573:Formycins / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006151:Guanosine / Q000737:chemistry*; D015151:Immunoblotting; D007288:Inosine / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000378:metabolism*; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D033703:Nucleoside Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D002352:Carrier Proteins; D005573:Formycins; C411852:LdNT2 protein, Leishmania donovani; D008024:Ligands; D008164:Luminescent Proteins; D033703:Nucleoside Transport Proteins; D015800:Protozoan Proteins; D006151:Guanosine; C034561:formycin B; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D007288:Inosine; D001120:Arginine