From the
We previously characterized a purine-specific
Na
Animal liver contains abundant 5-phosphoribosyl-1-pyrophosphate
amidotransferase, the first enzyme in de novo purine
biosynthesis, and rapidly incorporates
[
A major source of intracanalicular purine nucleosides is likely to
be the breakdown of nucleotides by ectonucleotidases, which co-localize
in bile canalicular membrane with the nucleoside
transporter
(3) . Both ectonucleotidases and the
Na
Nucleoside transport has become the focus of much
experimental work because of clinical use of nucleoside
analogues
(9, 10) . Based on substrate specificities,
Na
The goal of this study was to isolate the
cDNA encoding the Na
Poly(A)
Comparison with protein sequences in the data bases revealed that
several regions of SPNT share significant identity with a number of
bacterial membrane proteins, including three nucleoside transport
proteins (BSDNPOP_4, ECNUPC_1, and NUPC_ECOLI) and two hypothetical
43.4-kDa proteins (ECOHU47_52 and ECOHU47_55) of unknown function
(Fig. 4C). Four consensus sequences (EGSXFVFG,
VXSILYYLGLM, NEFVAY, and
SXXLXXFANFSSIGIXXG) were derived,
which are conserved in sequence and location, i.e. similar
space between these consensus sequences. Homology in these regions may
reflect common functions, such as facilitating cation or nucleoside
binding. We speculate that genes encoding mammalian and bacterial
nucleoside transporters may have evolved from the same evolutionary
precursors and subsequently underwent extensive evolutionary
divergence. The conserved regions may be critical for nucleoside
transport function and could be used in searching for additional
members of this family.
In summary, we have isolated a bile
canalicular Na
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
-nucleoside cotransport system in bile canalicular
membrane. The function of this transport system may be related to
conserving nucleosides and preventing cholestasis. We report here the
isolation of a cDNA encoding a Na
-dependent nucleoside
transporter from rat liver using an expression cloning strategy. The
substrate specificities and kinetic characteristics of the cloned
cotransporter are consistent with the properties of the
Na
-dependent, purine-selective nucleoside transporter
in bile canalicular membranes. The nucleotide sequence predicts a
protein of 659 amino acids (72 kDa) with 14 putative membrane-spanning
domains. Northern blot analysis showed that the transcripts are present
in liver and several other tissues. Data base searches indicate
significant sequence similarity to the pyrimidine-selective nucleoside
transporter (cNT1) of rat jejunum. Although these two subtypes of
Na
-nucleoside cotransporter have different substrate
specificities and tissue localizations, they are members of a single
gene family.
C]formate into purine nucleotides in vivo and in vitro(1) . Incorporation of
[
C]formate into bone marrow DNA purines is
depressed following partial hepatectomy, which suggests that the liver
is a major source of purine for salvage pathways
(1) . A
Na
-dependent purine nucleoside cotransporter system
has been localized to the bile canalicular membrane
(2) . One
function of this transporter is related to maintaining the nucleoside
concentration in hepatocytes and preventing nucleoside loss in bile.
-dependent purine nucleoside transport may be
important not only for purine salvage but also in regulating the effect
of ATP and adenosine on liver function. ATP and adenosine exert
multiple receptor-mediated effects on isolated
hepatocytes
(4, 5) , and it is unclear whether these
purinergic receptors are canalicular. Hypoxia releases ATP from human
erythrocytes and cardiac myocytes
(6, 7) and produces
canalicular cholestasis
(8) , suggesting that intracanalicular
ATP and/or its breakdown product, adenosine, may be increased in
hypoxia resulting in cholestasis through increased purinergic
receptor-mediated intracellular cAMP and Ca
. The
ectonucleotidases, purinergic receptors, and nucleoside transporter may
interact coordinately to conserve nucleoside and prevent cholestasis.
The cloning and expression of the canalicular
Na
-dependent purine nucleoside transport permits study
of its regulation and relation to ectonucleotidases and purinergic
receptors.
-dependent nucleoside transporters have been
classified into three subtypes: Cif (selective for purine nucleosides
and uridine)
(2, 11, 12) ; Cit (selective for
pyrimidine nucleosides and adenosine)
(13, 14) ; and Cib
(broadly specific for purine and pyrimidine nucleosides)
(15) .
It is not known whether these transporters belong to a single
structural family. Recently, two cDNA clones encoding putative
Na
-dependent nucleoside transporter proteins were
identified. One clone was isolated from rat jejunum and encoded a
nucleoside transporter with characteristics of the pyrimidine selective
(Cit) nucleoside transporter (cNT1). The transcript of cNT1 was
detected only in jejunum and kidney
(16) . The other clone
(SNST1) was isolated from rabbit kidney by hybridization with the
rabbit Na
-glucose cotransporter and encoded a
nucleoside transporter with broad specificity for purines and
pyrimidines. The transcript of SNST1 was detected only in kidney and
heart
(17) . The clones had no significant sequence homology.
Both transcripts were absent from liver and had different substrate
specificities when compared with the canalicular nucleoside transport,
suggesting that the Na
-dependent purine nucleoside
transporter (Cif) in rat liver is a distinct gene product with unique
structure and function.
-dependent purine nucleoside
transporter (SPNT)
(
)
from rat liver by expression
cloning in Xenopus laevis oocytes and determine whether SPNT
and cNT1 or SNST1 belong to the same family and are evolved from a
common evolutionary precursor. Based upon molecular characteristics of
these functionally distinct nucleoside transporters, we are now able to
study substrate binding sites, tissue-specific function, and
regulation, which will provide important information for drug design
and clinical application.
RNA Isolation and Poly(A)
Total RNA was extracted from
male Wistar rat liver and other tissues using the acid guanidine
thiocyanate/phenol/chloroform extraction method
(18) .
Poly(A)RNA
Selection and Fractionation
RNA was selected by oligo(dT)-cellulose
(Collaborative Research Inc.) chromatography and fractionated according
to size using sucrose gradient centrifugation
(19) .
X. laevis Oocytes and Na
Oocytes were dissected
from ovarian fragments of X. laevis, defolliculated manually,
and incubated in modified Barth's solution consisting of 73
mM NaCl, 1 mM MgCl-dependent
Nucleoside Transport Measurements
, 0.5 mM
CaCl
, 25 mM HEPES, 1 mM KCl, 100
µg/ml bovine serum albumin, 10 mM NaHCO
, and
50 µg/ml gentamicin at 18 °C. Nucleoside uptake into oocytes
was measured by a radiotracer technique
(20) . In brief, oocytes
were washed with a Na
-free buffer (100 mM
choline chloride, 2 mM KCl, 1 mM CaCl
, 1
mM MgCl
, and 10 mM HEPES-Tris (pH 7.5))
and then placed in a Na
(100 mM NaCl) or
sodium-free (100 mM choline chloride) buffer containing
[2,8-
H]adenosine (32.9 Ci/mmol, DuPont NEN).
After incubation for 1-120 min at 20 °C, adenosine uptake was
terminated by removing the incubation medium and washing the oocytes
with two 10-ml aliquots of ice-cold choline chloride buffer containing
1 mM unlabeled adenosine. Each oocyte was dissolved in 0.5 ml
of 10% (w/w) SDS. After addition of 5 ml of scintillation fluid,
oocyte-associated radioactivity was counted in a liquid scintillation
spectrometer (Beckman, LS 1801).
Library Construction and Clone Isolation
A
directional cDNA library was constructed in the plasmid vector pSPORT1
(SuperScript, Life Technologies, Inc.) using the size-fractionated
poly(A) RNA that gave rise to peak
Na
-dependent adenosine uptake. cDNA of 2-4 kb
was ligated into pSPORT1 plasmid, which was electroporated into
DH
cells. cRNA was transcribed in vitro using
T
RNA polymerase in the presence of Cap analog
(m
GpppG; Pharmacia Biotech Inc.) from pools of clones and
injected into oocytes. A pool that induced
Na
-dependent adenosine uptake was progressively
subdivided until a single clone with a cDNA insert of 2.9 kb was
isolated (SPNT).
Nucleotide Sequencing and Sequence Analysis
Both
strands of SPNT cDNA insert were sequenced by the dideoxy chain
termination method using the Sequenase DNA sequencing kit and protocol
(U. S. Biochemical Corp.). BLAST was used for data base search, and the
Genetics Computer Group sequence analysis software package was used to
analyze nucleotides and the deduced amino acid sequences.
Northern Blot Analysis
Total RNA from rat liver
and other tissues was separated on a 1% formaldehyde-agarose gel and
blotted onto a GeneScreen Plus membrane (DuPont NEN). The
HindIII-SalI (1958 base pairs) fragment of SPNT that
represented coding sequences for SPNT amino acid residues 1-562
was labeled with [P]dCTP (DuPont NEN) using
Klenow enzyme (Boehringer Mannheim). Hybridizations were performed at
42 °C in 6
SSPE, 50% formamide, 10
Denhardt's
solution, 1% SDS, 10% dextran sulfate, 100 µg/ml salmon sperm DNA.
The membranes were washed twice in 2% SSC, 0.5% SDS at room
temperature, twice at 1% SSC, 0.5% SDS at 45 °C, and once at 0.2%
SSC, 0.2% SDS at 65 °C.
RNA (mRNA) isolated from rat liver
gives rise to Na
-dependent adenosine uptake when
injected into Xenopus oocytes (Fig. 1A).
Significant expression of adenosine uptake was observed on day 1 and
increased progressively from day 1 to day 3 after mRNA injection.
Compared with endogenous transport activity (H
O injected or
uninjected), 13- and 30-fold increases in Na
-dependent
adenosine uptake were observed on day 1 and day 3, respectively, after
mRNA injection. Augmented Na
-dependent adenosine
uptake was mainly observed in oocytes injected with larger sizes of
mRNA (fractions 1-11) (Fig. 1B). Fractions
5-9 (2-4 kb) were pooled and used to construct a
directional cDNA library. To screen the cDNA library, we prepared sense
cRNA by in vitro transcription from pools of
1000 clones,
injected the cRNA into oocytes, and assayed expression of
Na
-dependent adenosine uptake. One positive pool was
progressively subdivided until a single clone with a cDNA insert of 2.9
kb was isolated. Na
-dependent adenosine uptake in
cRNA-injected oocytes increased nearly linearly within 120 min
(Fig. 2A). At cRNA doses above 1 ng, the expressed
transport activities were not linearly related to the amounts of cRNA
injected, suggesting that the translation machinery is saturable, which
may also explain why the expression level of the isolated clone is not
much larger than that of pooled RNA. Competition studies with different
purine and pyrimidine nucleosides showed that adenosine uptake was
almost completely inhibited by inosine, guanosine, and uridine and only
partially inhibited by thymidine and cytidine. SPNT activity was fully
inhibited by formycin B, partially inhibited by a purine analog Ara-A,
and not inhibited by dipyridamole or azidothymidine
(Fig. 2B). The kinetics of SPNT for adenosine and
thymidine were compared by examining the initial rates of uptake over a
range of concentrations of adenosine and thymidine. Uptake of both
nucleosides by the oocytes expressing SPNT was saturable. SPNT has a
K
of 6 µM for adenosine and
13 µM for thymidine; the V
is 457
fmol/oocyte/min for adenosine and 13 fmol/oocyte/min for thymidine.
These data indicate that SPNT is the functional
Na
-dependent purine nucleoside transporter (Cif type)
previously characterized in rat liver canalicular plasma
membrane
(2) .
Figure 1:
A, time course
of expression of adenosine uptake in X. laevis oocytes.
Oocytes were either not injected (,
) or injected with 50
ng of rat liver poly(A)
RNA (mRNA) (
,
).
Uptake of 15 µM [
H]adenosine was
measured at room temperature (20 °C) in the presence of 100
mM NaCl (
,
) or 100 mM choline chloride
(
,
). B, [
H]adenosine
uptake in oocytes after 3 days of injection with 50 nl of
size-fractionated poly(A)
RNA, in the presence of NaCl
(
) or choline chloride (
). T, total RNA (50
ng/50 nl); M, mRNA (50 ng/50 nl); 1-24, pools
of size-fractionated poly(A)
RNA (10-20 ng/50
nl); -, uninjected oocytes. Uptake values are means ± S.E.
obtained with 8-10 oocytes. If S.E. bars are not visible
they are smaller than the symbols.
Figure 2:
A, time course of
[H]adenosine uptake in cRNA-injected oocytes.
Oocytes were not injected (
,
) or injected with 10 ng of
synthetic RNA (cRNA) (
,
) transcribed from the SPNT clone.
Three days after injection, 15 µM
[
H]adenosine uptake was determined at room
temperature (20 °C) over 120 min in the presence of 100 mM
NaCl (
,
) or choline chloride (
,
).
B, effects of nucleoside transport inhibitors and nucleoside
analogues on adenosine uptake in cRNA-injected oocytes. Oocytes were
treated as described in A. 15 µM
[
H]adenosine uptake was determined at 20 °C
in the presence and in the absence (control) of the indicated 1
mM various compounds (
, NaCl;
, choline
chloride). C and D, initial rates of
[
H]adenosine (C) and
[
H]thymidine (D) uptake was measured
over 1 min using a range of concentrations of 1-1500
µM for adenosine and 1-300 µM for
thymidine. Na
-specific uptake was determined by
subtracting the value in the presence of choline chloride (
)
from the value in the presence of NaCl (
). K and
V
were determined from a double-reciprocal plot
(not shown) of these data. Uptake values are mean ± S.E.
obtained with 8-10 oocytes. DPA, dipyridamole;
AZT, azidothymidine.
We examined the tissue distribution of SPNT
expression by Northern blot analysis (Fig. 3). A 2.9-kb
transcript was detected in liver, jejunum, spleen, and heart.
Additionally, two minor bands corresponding to RNAs of sizes
6 and
1 kb were seen in spleen, jejunum, heart, brain, and skeletal
muscle. The function of the latter two transcripts is unknown. The
multiple transcripts and tissue expression may relate to diverse
function of purine nucleosides, especially adenosine, in various
tissues. The transporter may be involved in regulation of purinergic
receptor-mediated nucleoside effects in addition to salvaging
nucleosides. In contrast, cNT1 was only detected in intestine and
kidney, where it may be mainly involved in nucleoside absorption and
reabsorption in these two tissues
(16) .
Figure 3:
Northern blot analysis of SPNT transcript.
Total RNAs (25 µg/lane) from kidney (K), spleen
(S), jejunum (J), heart (H), brain
(B), muscle (M), lung (Lu), and liver
(L) tissues were separated by agarose gel electrophoresis and
hybridized with the P-labeled SPNT probe as described
under ``Experimental Procedures.'' Equal loading was checked
by rehybridization with the rRNA probe.
The 2907-base pair
sequence of SPNT cDNA contains a single long open reading frame of 1980
nucleotides encoding a protein of 659 amino acid residues with an
estimated relative molecular mass of 72 kDa
(Fig. 4A). Hydropathy analysis (Fig. 4B)
revealed 14 putative membrane-spanning segments. There are five
possible N-glycosylation sites (Asn-439, -539, -603, -606, and
-625), one ATP/GTP binding motif (GXXXXGKT) in the N terminus,
and several consensus sites for protein kinases A (RXS) and C
(SXR, SXK, and TXR) phosphorylation in both
termini (Fig. 4A). These data suggest that SPNT may be
regulated by intracellular ATP/GTP and protein kinases A and C.
Figure 4:
A, predicted amino acid sequences of the
cDNA, SPNT, encoding the Na-dependent purine
nucleoside transporter. Consensus sites for protein kinase A (#) and C
(*) phosphorylation and five potential N-linked glycosylation
sites (underscored) are shown. B, hydropathy plot
using the Kyte-Doolittle method (22) with a window size of 8 amino
acids showing putative transmembrane segments 1-14. C,
alignment of relevant regions of amino acid sequences of several
proteins that share significant homology with SPNT. The locations of
these amino acid residues in their respective proteins are indicated by
numbers flanking the displayed sequence. Proposed consensus
sequences and gaps are displayed in the bottomline,
suggesting protein sequence conservation. Sequence data are shown for
SPNT, cNT1, ECOHU47_55 yeiM (Escherichia coli) hypothetical
43.4-kDa protein, ECOHU47_52 yeiJ (E. coli) hypothetical
43.4-kDa protein, BSDNPOP_4 (Bacillussubtilis)
pyrimidine nucleoside transport protein, ECNUPC_1 NupC (E.
coli), and NUPC_ECOLI nucleoside permease
NUPC.
An
important feature of SPNT is structural similarity (64% identity at the
amino acid level) to cNT1 and differences in substrate specificities
and tissue distribution. The most divergent regions are in the N- and
C-terminal domains, which may be important in tissue-specific
regulation. For example, the ATP/GTP binding motif is present in the N
terminus of SPNT but not in cNT1. The putative protein kinase A and C
phosphorylation sites are also different in locations and numbers.
Although the transmembrane regions have similar sequences over most of
their length, a number of short stretches of sequence with different
amino acids are found. These differences may be sufficient to confer
substrate specificity of subtypes of nucleoside transporter as reported
for adenosine receptors
(21) . The structural similarity of these
two proteins suggests that they are members of the same gene family. In
contrast, there is no significant homology between SPNT and SNST1.
purine nucleoside co-transporter by
expression cloning in Xenopus oocytes. Transcripts were
present in multiple tissues. The structural features found in SPNT,
cNT1, and bacterial nucleoside transporters suggest that they are
members of a single nucleoside transporter superfamily. Several
consensus regions that may be important in transport functions were
identified.
/EMBL Data Bank with accession number(s) U25055.
-dependent purine nucleoside transporter; kb,
kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.