Cloning of a novel inosine-guanosine transporter gene from Leishmania donovani by functional rescue of a transport-deficient mutant.
Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 re-established their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K(m) = 0.3 micrometer) and guanosine (K(m) = 1.7 micrometer) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.
Carter
N S
NS
Department of Biochemistry, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Drew
M E
ME
Sanchez
M
M
Vasudevan
G
G
Landfear
S M
SM
Ullman
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005573:Formycins / Q000494:pharmacology; D006151:Guanosine / Q000378:metabolism*; D007288:Inosine / Q000378:metabolism*; D007700:Kinetics; D007893:Leishmania donovani / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins; D009865:Oocytes / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014162:Transfection; D014981:Xenopus
D002352:Carrier Proteins; D005573:Formycins; C411852:LdNT2 protein, Leishmania donovani; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D015800:Protozoan Proteins; D006151:Guanosine; C034561:formycin B; D007288:Inosine