trans

204 PURINES AND PYRIMIDINES
physiological purine concentrations nucleobase
transport is repressed. Purine nucleoside
transport also is affected by conditions
of purine stress, although to a much lesser
degree. Recently, an ENT homolog, LmNT3,
was identified within the L. major genome
database. LmNT3 encodes a 501 amino acid
ENT-type transporter that has 33% identity to
LdNT1 and 35% to LdNT2. When functionally
characterized in Xenopus oocytes, LmNT3 was
found to be a high-affinity transporter for adenine,
hypoxanthine, guanine, and possibly
guanosine. Surprisingly, LmNT3 does not
transport xanthine, suggesting that at least one
other nucleobase transporter gene is encoded
within the leishmanial genome.
It is entirely feasible that other purine and
pyrimidine transporters are present within the
leishmanial genome. Some of the major purine
salvage enzymes in Kinetoplastida are compartmentalized
in the glycosome, probably
requiring the translocation of purine bases
into this unique organelle. Localization studies
with Green Fluorescent Protein-coupled transporters
have shown that these transporters
localize primarily to the parasite plasma
membrane (and possibly to the flagellum in
the case of LdNT2). In addition to these other
intracellular transporters, it is possible that
different nucleoside and nucleobase transporters
are expressed within the amastigote
life stage of the parasite. Recent studies on
intact L. donovani amastigotes revealed two
distinct adenosine transport activities, an
LdNT1-like activity, and a second activity that
was inhibited by the purine nucleosides inosine
and guanosine, but not by pyrimidines.
Whether LdNT1 and LdNT2 are expressed
in amastigotes has not been determined,
although a partial cDNA sequence from
L. major amastigotes with high homology to
LdNT2 has been deposited into the GenBank
database.
As mentioned above, purine transport is
upregulated in several species of Trypanosomatidae
under conditions of purine depletion.
Transport is also modulated in Leishmania by
the TOxic nucleoside Resistance protein (TOR),
named because it was first identified in
L. donovani selected for resistance to the toxic
adenosine analog, tubercidin. TOR comprises
some 478 amino acids and is similar to the
mammalian transcriptional activator OCT-6.
When overexpressed in Leishmania TOR
represses purine nucleoside, and most significantly
nucleobase, transport. The mechanism
for this phenomenon is unknown. TOR
can also function as a transcriptional activator
in yeast, although it is notable that transcriptional
enhancers have not been described for
Leishmania.
Trypanosoma cruzi
Very little is known about nucleoside and
nucleobase transport into T. cruzi. Most of
our knowledge is derived from studies on
tubercidin-resistant epimastigote-stage parasites.
These have shown that, like Leishmania
and Crithidia, these parasites possess an
adenosine–thymidine transporter, and possibly
other adenosine, inosine and uridine transporters.
Expression Site Tagged (EST) sequences
homologous to LdNT1 and LdNT2 have been
found for T. cruzi epimastigotes.
Trypanosoma brucei and other African
trypanosomes
Unlike other kinetoplastids, African trypanosomes
contain a plethora of purine and
pyrimidine transporters with overlapping substrate
specificities that are stage-specifically
expressed in the procyclic (insect stage) and
bloodstream (mammalian infectious stage)
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA