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Yeast 14, 1257–1265 (1998)
Cloning of the Candida albicans Nucleoside Transporter
by Complementation of Nucleoside Transport-deficient
University of North Dakota School of Medicine, Department of Biochemistry and Molecular Biology, Grand Forks,
ND 58202, U.S.A.
The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces
cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was
sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine,
cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was
different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded
for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli
nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The
GenBank accession number for the Candida NUP gene is AF016246. 1998 John Wiley & Sons, Ltd.
  — Candida albicans; nucleoside transport
In contrast to Saccharomyces cerevisiae, which
normally appears to lack a detectable purine
nucleoside transport capability, Candida albicans
has been found to possess not only pyrimidine
nucleoside transport capability but purine nucleoside transport capability as well. Rao et al. concluded from competitive inhibition experiments
that a purine nucleoside permease existed which
transported adenosine, inosine and thymidine, but
not uridine (Rao et al., 1983). Uridine transport
was not significantly affected by an excess of
adenosine or thymidine, suggesting that it was
transported by a separate permease. Fasoli and
Kerridge (1990) also detected at least two separate
nucleoside permeases. However, based on similar
competition experiments, they concluded that
uridine permease also transported cytidine and
adenosine, but not thymidine. In support of this
model they noted that the uracil permease mutant
6F1 also exhibited reduced ability for adenosine
transport. Thymidine transport was detected in
Candida but it did not compete with uridine
*Correspondence to: S. Detke.
CCC 0749–503X/98/141257–09 $17.50
1998 John Wiley & Sons, Ltd.
and they surmised that a separate thymidine
transporter existed.
To resolve these differences, I sought to clone
and express individual Candida nucleoside permease genes in Saccharomyces. The absence of
purine nucleoside transport activity in wild-type
Saccharomyces suggested that the specificity of
individual nucleoside permeases could be more
clearly analysed if only a single transporter were to
be present. In addition, these Candida permeases
warranted further study, as they have been implicated as virulence factors in the pathogenicity of
this organism (Fasoli et al., 1990). Normally a
commensal, Candida has been increasingly found
to be an opportunistic pathogen in the immunecompromised. Individuals treated with immune
suppressants, for example, and those with a
reduced immune system (e.g. HIV-infected individuals) exhibited an increased frequency of infection
by this organism (Scully et al., 1994).
Growth of yeast
Saccharomyces cerevisiae BWGI-7a (a, ade1,
his4, leu2, ura3) and Candida albicans SGY126
Received 8 August 1997
Accepted 5 June 1998
. 
Figure 1. Removal of DNA flanking the Candida nucleoside permease gene. The nested deletions spanning the left and
right flanks were constructed as described in the Methods section. BWGI-7a transformed with each truncation product
was assessed for its ability to grow on selective YNB plates supplemented with either adenosine or adenine as the sole
purine source. RCE, colony forming efficiency on minimal medium plates containing adenosine relative to minimal
medium plates containing adenine. There were too many colonies to accurately count on the plates testing the right
flank, so small areas selected at random on these plates were analysed. There was growth on all minimal medium plates
containing adenine. Restriction sites in the vector DNA flanking the Candida insert: E, EcoRI; S, SacI; B, BamHI; X,
XbaI; P, PstI; H, HindIII. The heavy bars represent the Candida insert. Vector DNA other than the small multi-cloning
sites flanking the insert is not shown.
(ade) were grown in 1% (w/v) Bacto yeast extract,
2% (v/v) Bacto peptone, 2% (w/v) glucose (i.e.
YPD) at 30C. SGY126 was purchased from the
American Type Culture Collection (Manassas,
VA). Transformed yeast were propagated in minimal medium (yeast nitrogen base, Difco), 0·5%
(w/v) glucose and 50 ìg/ml leucine and histidine,
hereafter referred to as YNB) with either adenosine or adenine at 50 ìg/ml for the purine source, as
indicated in the text. Adenine was used for routine
propagation of transformed cells instead of adenosine to reduce the possibility of acquiring revertants of Saccharomyces for nucleoside transport,
or acquiring mutants which degrade extracellular
adenosine to adenine and transporting this base
for survival and growth. Uracil was omitted in the
minimal medium to prevent Saccharomyces from
losing the plasmid in the absence of selective
pressure with adenosine.
S. cerevisiae were transformed overnight via an
extended PEG–lithium acetate protocol (Elble,
1992). Transformed yeast carrying the Candida
albicans nucleoside permease gene were selected on
agar plates containing YNB, 0·5% (w/v) glucose
and 50 ìg/ml leucine, histidine and adenosine.
1998 John Wiley & Sons, Ltd.
Removal of DNA flanking the Candida nucleoside
permease gene
The YEp352 vector carrying the 6·5 kb insert
from pCNT (i.e. clone Ba in Figure 1) was digested
with BamHI and SacI to yield, respectively, an
exonuclease III-sensitive site proximal to the left
flank of the insert, and a distal resistant site
protecting the vector from exonuclease III digestion. Nested deletions covering the left flank were
created with exonuclease III and assessed for their
ability to enable BWGI-7a to grow on selective
YNB plates with either adenosine or adenine as the
purine source (Sambrook et al., 1989). Clone Bd
was digested with XbaI and PstI to yield exonuclease III sensitive and resistant termini prior to
exonuclease III digestion.
DNA sequencing
The insert in pNUP was excised with EcoRI and
HindIII and cloned into the corresponding sites of
pBluescript (Stratagene, La Jolla, CA). Nested
deletions were created with exonuclease III and
sequenced by the dideoxy method (Sambrook
et al., 1989; Sanger et al., 1977). Both strands were
Yeast 14, 1257–1265 (1998)
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RNA analysis
RNA was isolated from spheroplasts with
Ambion’s ToTally RNA Kit (Ambion Inc.,
Austin, TX) and electrophoresed in a MOPS–
formaldehyde agarose gel (Rueger et al., 1996). To
generate the appropriate hybridization probes, a
portion of the cloned C. albicans nucleoside permease was amplified with Pfu (Stratagene, La
Jolla, CA) and the primers taatacgactcactatagg
TCATGACAAGATTGG and aattaccctcactaaagg
AAGCAGTATCAAATG (the lower case letters
are the sequences for the T7 and T3 promoters,
respectively). A portion of the S. cerevisiae actin
open reading frame YFL039C (Research Genetics
Inc., Huntsville, AL) was amplified with Pfu and
the primers: taatacgactcactataggTGTTCCCAAG
TATTG and aattaccctcactaaaggTTAGAAACAC
TTGTTG (the lower case letters are again the
sequences for the T7 and T3 promoters, respectively). Antisense RNA was synthesized by T3
polymerase with the MAXIscript T7/T3 kit
(Ambion) and digoxigenin-UTP (Boehringer
Mannheim Corp., Indianapolis, IN). Hybridization was conducted at 60C in the phosphate–SDS
buffer described by Engler-Blum et al. (1993) but
post-hybridization washes were in 0·1SSC, 0·1%
SDS. Hybrids were detected with anti-digoxigenin
antibody–horseradish peroxidase, as described in
Boehringer Mannheim’s Dig labelling and
detection kit manual, with SuperSignal NA
(Pierce, Rockford, IL). The light emitted in
this chemiluminescent assay was captured on
X-ray film.
Purine transport assay
An overnight culture was diluted with two
volumes of medium and incubated at 30C for a
further 2 h. The cells were then centrifuged at
2000g for 5 min, washed twice with YNB, 0·5%
glucose, and resuspended in YNB, 0·5% glucose, at
2108 cells/ml. Transport was initiated by adding
100 ìl of cells to 100 ìl of YNB/glucose with 2 ì
H-purine (or pyrimidine) placed on top of a
200 ìl pad of dibutyl phthalate. Transport was
terminated by centrifugation at 12,000g for 15 s.
The unincorporated substrates in the aqueous
phase over the oil were removed by aspiration.
After the upper surface of the oil was rinsed twice
with 500 ìl water, the oil was removed by aspiration and the pellet resuspended with 200 ìl water.
Radioactivity in the resuspended pellet was determined by liquid scintillation spectrophotometry
with CytoScint (ICN, Costa Mesa, CA).
1998 John Wiley & Sons, Ltd.
Cloning of the Candida albicans nucleoside
Saccharomyces cerevisiae ade mutants were unable to grow on defined medium supplemented
with purine nucleosides because this yeast normally lacked transport capability for these substrates. The Candida albicans ade mutant SGY126,
on the other hand, grew equally well on minimal
medium supplemented with either adenosine or
adenine, presumably because it possessed the
required adenine and adenosine permeases (growth
data not shown: Fasoli and Kerridge, 1990; Polak
and Grenson, 1973; Rao et al., 1983). Because
Candida genes can complement genetic defects in
Saccharomyces, I sought to clone the Candida
nucleoside permease(s) by selecting for growth of
transformed Saccharomyces on minimal medium
supplemented with adenosine. Saccharomyces
BWG1-7a could not synthesize purines de novo, so
they could grow on minimal medium supplemented with adenosine if they acquired the Candida equivalent of the ade 1 gene or the Candida
nucleoside permease.
Saccharomyces were transformed with a Candida albicans genomic library carried in pYSK35
and selected on minimal medium plates supplemented with adenosine. Two hundred colonies
were obtained from 16,000 transformed yeast.
Approximately 80% of these colonies were very
small and were not considered further. Twelve of
the largest colonies were expanded and their plasmids transferred to new yeast to determine
whether growth under the selection conditions
were transferable. Only one of these plasmids
yielded transformed Saccharomyces, which grew
on the minimal plates supplemented with adenosine as well as on minimal plates in which adenosine
was replaced with adenine. This plasmid will
hereafter be referred to as pCNT.
The Candida insert in pCNT was 6·5 kb. To
eliminate extraneous flanking DNA, the insert was
first excised with XbaI and moved into the corresponding site in YEp352 (clone Ba in Figure 1).
The left flank of the insert was then sequentially
reduced with exonuclease III to create nested deletions and each truncated clone was tested for its
ability to enable growth of transformed BWG1-7a
on minimal medium plates containing adenosine as
the purine source. Clone Bd had approximately
2·5 kb of flanking DNA removed, yet still enabled
BWGI-7a to grow as well on minimal medium
Yeast 14, 1257–1265 (1998)
plates supplemented with adenosine, as compared
to growth on minimal medium plates in which
adenosine was replaced with adenine. The adenine
would have been transported by the endogenous
FCY2 purine permease of Saccharomyces (Weber
et al., 1990). The next smallest clone, Be, no longer
conferred upon BWGI-7a the ability to use
exogenous adenosine for growth, suggesting that
part of the permease gene per se was removed. The
right flank was treated in a similar manner, yielding clone Xd, the smallest of these exonuclease III
truncation products, which still permitted growth
of BWGI-7a on selective plates supplemented with
adenosine with the same efficiency as on selective
plates in which adenosine was replaced with
adenine. Clone Xb will hereafter be called pNUP
and contained an insert of 1·9 kb.
Sequence of the Candida nucleoside transporter
The DNA sequence of the insert in pNUP was
analysed with DNASIS (Hitachi Software Co. Ltd,
San Bruno, CA) and a single open reading frame
was identified. The nucleotide and amino acid
sequence of the Candida purine nucleoside permease is shown in Figure 2. The NUP gene product coded for a protein of 407 amino acids in size
with a molecular weight of 44830. A search
through the BLOCKS and PRINTS databases
with the BLOCKS search program did not yield
any significant local similarities to either identify
the Candida gene or to determine its function
(Henikoff and Henikoff, 1994). An analysis of the
amino acid sequence with PSORT (prediction of
protein sorting signals and localization sites in
amino acid sequences; WWW address, http://, however, indicated that the
N-terminus has the properties of a signal peptide,
which suggested a membrane location for this
protein (von-Heijne, 1988).
A search of the non-redundant DNA, SwissProt
and Saccharomyces databases of the National
Center for Biotechnology Information with the
Candida nucleoside permease sequence using the
BLASTp and BLASTn programs did not yield any
matches of high enough probability to be informative. In addition to these general searches, the
Candida sequence was compared directly with the
sequences of the purine nucleoside transporters
found in GenBank. There were no significant
regions of homology at the amino acid level
between the Candida permease and the E. coli
nupC permease (accession number X74825), E. coli
1998 John Wiley & Sons, Ltd.
. 
nupG permease (accession number X06174),
human and rat equilibrative nucleoside transporters 1 and 2 (accession numbers U81375,
AF015304, AF015305, AF029358 and AF034102),
Giardia lamblia permease (accession number
L11576) or the human and rat sodium-dependent
nucleoside transporter (accession numbers U84392
and U10279). These data would suggest that either
the Candida permease diverged greatly from these
other permeases over evolutionary time, or that
convergent evolution has occurred to yield a
permease of similar function.
A search through the Candida NUP DNA
sequence with the DNA motif finder in DNASIS
suggested that the TATA box was located between
nucleotides 209 and 219. At the opposite end of the
insert, the three components required for polyadenylation in Saccharomyces were found (Guo and
Sherman, 1995). Three perfect copies of the hexanucleotide efficiency element, TATATA, were
found in a tandem repeat which would function
additively (reviewed in Wahle and Keller, 1996).
Downstream of this was the hexanucleotide positioning element TATAAA. The Candida positioning element differed by one nucleotide from the
highly conserved positioning element AATAAA
found in other eukaryotes, but Guo and Sherman
showed that an A to T mutation at the first
nucleotide yielded a positioning element which
functioned just as efficiently as the canonical
sequence (Guo and Sherman, 1995). The potential
polyadenylation site, TAAA, was found 25 nucleotides downstream of the positioning element. Not
only were the sequences of these Candida elements
almost completely identical to the corresponding
Saccharomyces elements, but the spacing between
them was within the limits of the spacing observed
in Saccharomyces.
To verify that this cloned gene was expressed, a
Northern blot was probed with a digoxigeninlabelled antisense RNA directed against the 3 end
of the open reading frame found within this insert.
S. cerevisiae transformed with pNUP expressed as
RNA of approximately 1·4 kb, which hybridized
to the probe, but yeast transformed with the
parental vector YEp352 did not (Figure 3). The
absence of a hybridization signal in lane 1 was due
to the absence of homologous RNA and not
degraded RNA, as the actin probe hybridized to
both samples. The size of the NUP mRNA, as
determined by agarose gel electrophoresis, was in
close agreement to the estimated 1·5 kb size based
on the distance from the presumptive TATA
Yeast 14, 1257–1265 (1998)
     
Figure 2. Nucleotide and amino acid sequence of the Candida purine nucleoside permease. Bold
nucleotides between 209 and 219, potential TATA box. Polyadenylation components: hexanucleotide
efficiency elements enclosed in boxes; hexanucleotide positioning element identified by single underline;
polyadenylation site identified by double underline. The GenBank accession number is AF016246.
1998 John Wiley & Sons, Ltd.
Yeast 14, 1257–1265 (1998)
. 
sequences and not by the distance from the TATA
element (Chen and Struhl, 1985).
Figure 3. Expression of the C. albicans nucleoside transporter
in S. cerevisiae; 10 ìg of total RNA was analysed, as described
in the Methods section. The yeasts were grown on minimal
medium supplemented with adenine. Lane 1, RNA from
YEp352-transformed S. cerevisiae; lane 2, RNA from pNUPtransformed S. cerevisiae. Top panel, C. albicans NUP hybridization probe; bottom panel, S. cerevisiae actin hybridization
element to the potential polyadenylation site plus a
60 poly-A tail (Groner et al., 1974). The initiation
site cannot be determined by a visual inspection of
the nucleotide sequence, as mRNA initiation sites
in Saccharomyces are determined by specific
Nucleoside transport in pNUP transformed
Although the sequence information did not yield
the identity of the Candida NUP gene, the following transport experiments indicated that the gene
coded for a Candida purine nucleoside permease.
The ability to transport purines was assessed by
measuring uptake over intervals up to 60 s. Normally, transport would have been measured over
intervals less than 30 s to minimize post-transport
metabolism, but Saccharomyces had a low-level,
time-dependent, saturable purine nucleoside binding capability which masked purine nucleoside
transport over very short intervals (Figure 4).
pNUP-transformed Saccharomyces were able to
accumulate adenosine and guanosine over this test
period at an accelerated linear rate, whereas
YEp352 transformed yeast were not. The Candida
permease apparently was specific for nucleosides,
as Saccharomyces transformed with pNUP did not
accumulate adenine at a significantly greater rate
than did the control YEp352 transformed yeast
(Figure 4A).
Fasoli and Kerridge (1990) found that a large
excess of cytosine and adenosine but not thymidine
Figure 4. Accumulation of adenine, adenosine and guanosine by YEp352 and
pNUP-transformed Saccharomyces. The accumulation of labelled test compound
over a 60 s interval by log phase cells was assessed as described in the Methods
section. The data consisted of the mean and standard deviation from three
experiments, except for guanosine uptake, which was the mean and range from
two experiments. (A) Adenine; (B) adenosine; (C) guanosine. Open circles,
YEp352-transformed BWGI-7a; filled circles, pNUP-transformed BWGI-7a.
1998 John Wiley & Sons, Ltd.
Yeast 14, 1257–1265 (1998)
     
Figure 5. Adenosine accumulation in the presence of a large excess of competing nucleosides to identify the
substrates for the purine nucleoside permease. The accumulation of adenosine over a 60 s period was assessed in the
presence of a 500-fold molar excess of unlabelled nucleoside. The inhibitory properties of dipyridamole at 200 ì and
NBMPR at 20 ì were also assessed. The data comprised the mean and standard deviation from three experiments.
reduced the transport of uridine and concluded
that the Candida nucleoside permease transported
adenosine and pyrimidine nucleosides except for
thymidine. It was assumed that competition
between two substrates for a common carrier
should result in a reduced accumulation of the
labelled test compound at a lesser concentration.
To determine whether this broad specificity Candida transporter was the gene cloned, the inhibitory effect of a large excess of competing
nucleoside on adenosine accumulation was accessed. An excess of unlabelled adenosine and
guanosine saturated the permease and reduced its
capacity to transport radiolabelled adenosine, as
expected for a purine nucleoside transporter (Figure 5). A 500-fold molar excess of uridine and
cytidine, on the other hand, had no effect on the
ability of the yeast to accumulate adenosine, suggesting that this purine nucleoside permease did
not transport these pyrimidine nucleosides. The
1998 John Wiley & Sons, Ltd.
absence of cytidine and uridine transport capability by this permease was also supported by an
earlier observation, showing that Saccharomyces
transformed with a random clone from the original
library in pYSK35 transported uridine as well as
did pCNT transformed cells. If the Candida permease had been able to transport these pyrimidines, pCNT transformed yeast should have
shown greater transport capability as compared
to the random clone, because they carried more
copies of the Candida nucleoside permease gene
than of the endogenous Saccharomyces pyrimidine
nucleoside permease. The YSK35 vector carried
the 2 ìm DNA for replication, rendering it a high
copy number plasmid in yeast (Schneider and
Guarente, 1991).
Alternatively, the Candida permease may have
had a much lower affinity for these two pyrimidine
nucleosides as compared to purine nucleosides,
and even a 500-fold molar excess may not have
Yeast 14, 1257–1265 (1998)
. 
Table 1. Growth of YEp352 and pNUP transformed Saccharomyces in the presence of
purine nucleoside transport inhibitors.
98 2
97 7
The cells were grown for one to two days at 30C in YNB supplemented with glucose, histidine,
leucine, the purine indicated above at 25 ìg/ml and dipyridamole at 200 ì or NBMPR at 20 ì. At
the end of the test period, cells were counted with a haemocytometer under phase–contrast
microscopy. Control cells grown in the absence of either dipyridamole or NBMPR were set at 100%
growth. The data are the growth relative to the control cultures and are the meanstandard deviation
of four separate experiments. YEp352 transformed cells do not grow with adenosine as the sole purine
been sufficient to compete with adenosine for
transport. If this was indeed the case, the cloned
Candida nucleoside permease would be different
from that described by Fassoli and Kerridge
(1990). Fassoli and Kerridge observed an inhibitory effect of cytosine and uridine on adenosine
transport at a much lower ratio of unlabelled to
labelled nucleosides than used in my experiments.
In contrast to uridine and cytidine, a 500-fold
molar excess of thymidine exhibited a slight ability
to reduce adenosine transport. Inhibition was only
20%, suggesting that the NUP gene product may
have a much higher affinity for purine nucleosides.
Attempts to verify thymidine transport by the
NUP permease were unsuccessful, as the accumulation was too low to measure accurately (data not
shown). Fasoli and Kerridge had also noted that
thymidine was transported very poorly by Candida
albicans, indicating that a different fungal environment was not the cause for the poor transport of
thymidine by these transformed Saccharomyces
(Fasoli and Kerridge, 1990). The apparent ability
of thymidine but not uridine to reduce adenosine
transport suggested that the cloned Candida nucleoside permease was similar to that described by
Rao et al. (1983).
The uptake of adenosine by the NUP gene
product was inhibited 100% with dipyridamole at
200 ì and 80% by NBMPR at 20 ì, two mammalian transport inhibitors (Figure 5). These two
compounds did not inhibit adenosine transport by
killing the yeast, as they had no inhibitory effect on
the growth of YEp352- or pNUP-transformed
Saccharomyces on minimal medium supplemented
with adenine (Table 1). Dipyridamole and
1998 John Wiley & Sons, Ltd.
NBMPR did, however, inhibit the growth of
pNUP-transformed Saccharomyces on minimal
medium supplemented with adenosine. The inhibition by both compounds was statistically significant, as judged by Student’s t-test (P<0·02). A
greater inhibition of growth by these two compounds was expected, based on the larger inhibition on transport observed in Figure 5.
Metabolism or breakdown of these inhibitory
compounds over this extended incubation period
may account for the difference between the
observed and expected results.
The transformed yeast incubated with NBMPR
in the presence of adenine grew slightly faster than
the control cultures. The stock solution of
NBMPR was dissolved in phosphate buffer, which
would have enriched the minimal medium with
additional phosphate and could have promoted
faster growth.
The Candida albicans genomic library was created
by Yigal Koltin and obtained from Mary Fling
(Wellcome Research Laboratories, Burroughs
Wellcome Co., Research Triangle Park, NC). Saccharomyces cerevisiae BWG1-7a was obtained
from D. M. Becker (MIT, Cambridge, MA). The
research was supported by NSF EPSCOR, Grant
No. OSR-9452892.
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Yeast 14, 1257–1265 (1998)
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