(Received for publication, January 4, 1996; and in revised form, February 7, 1996)
From the
A partial mouse cDNA was isolated by its ability to functionally
complement a thymidine transport deficiency in plasma membranes of the
yeast, Saccharomyces cerevisiae. The full-length cDNA encoded
a previously unidentified 27-kDa protein (mouse transporter protein
(MTP)) with four predicted transmembrane-spanning domains. MTP mRNA was
detected in cells of several mammalian species, and its predicted
protein sequence exhibited near identity (98%) with that of a human
cDNA (HUMORF13). MTP and its homologs evidently reside in an
intracellular membrane compartment because a protein (about 24 kDa)
that was recognized by MTP-specific antibodies was observed in a
subcellular fraction of rat hepatocytes enriched for Golgi membranes.
Deletion of the hydrophilic C terminus of MTP, which encompassed two
putative signal motifs for intracellular localization
(Tyr-X-X-hydrophobic amino acid), allowed expression
of recombinant protein (MTPC) in plasma membranes of Xenopus
laevis oocytes. MTP
C-expressing oocytes exhibited greater
fragility than nonexpressing oocytes, and those that survived the
experimental manipulations were capable of mediated uptake of
thymidine, uridine, and adenosine. Thymidine uptake by
MTP
C-expressing oocytes was inhibited by thymine and dTMP. MTP may
function in the transport of nucleosides and/or nucleoside derivatives
between the cytosol and the lumen of an intracellular membrane-bound
compartment.
Mammalian cells normally possess the capacity for mediated
translocation of nucleosides across their plasma membranes (for review
see (1) ). These nucleoside-selective transport (NT) ()processes function in the salvage of extracellular
nucleosides for energy metabolism and nucleic acid synthesis and play a
central role in regulating extracellular levels of adenosine, uridine,
and other naturally occurring nucleosides. NT processes also play an
important role in cellular uptake of nucleoside drugs. Seven
functionally distinct NT processes have been identified in plasma
membranes of mammalian cells, and it is likely that more exist.
Characterization of the proteins responsible for NT processes has been
hindered by their low abundance in plasma membranes. The identities of
three transporters (SNST1 of rabbit kidney, cNT1 of rat intestine, and
SPNT of rat liver) that mediate functionally distinct,
Na
-dependent NT processes have recently been
determined by expression cloning in oocytes of Xenopus
laevis(2, 3, 4) . Although cNT1 and SPNT
are members of the same transporter family, the absence of any sequence
homology between SNST1 and either cNT1 or SPNT suggests that NT
proteins of mammalian cells likely comprise a structurally diverse
group of transporter proteins.
The present study was undertaken to isolate cDNAs encoding equilibrative NT proteins of mammalian cells by functional expression in the yeast, Saccharomyces cerevisiae. Because it shares many cellular pathways with higher eukaryotes, S. cerevisiae has been used for identification of cDNAs encoding a variety of proteins from higher organisms, including several plasma membrane transporter proteins(5, 6, 7) . Using a phenotypic complementation strategy for isolation and molecular cloning of a cDNA, we have identified a unique transporter protein of mouse leukemia L1210 cells that evidently resides in intracellular membranes where it transfers nucleosides (and/or nucleoside metabolites) between the cytosol and a membrane-associated compartment(s). This protein, which appears to be widely distributed among mammalian species, was localized to intracellular membranes by the reactivity of native protein with MTP-specific antibodies, and its nucleoside transport activity was demonstrated by functional expression of a truncated recombinant transporter protein in X. laevis oocytes.
The cDNA inserts were manually sequenced (19) using a Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.) or by Taq DyeDeoxy terminator cycle sequencing with an Applied Biosystems Model 373A DNA Sequencer (DNA Sequencing Laboratory, Department of Biochemistry, University of Alberta). Deduced nucleotide and predicted protein sequences were compared with the sequence data bases of the National Center for Biotechnological Information and the Sequence Analysis Software GCG program (Genetics Computer Inc.).
Immunoblotting was performed on protein samples that had been solubilized under reducing conditions, separated on 12% SDS-polyacrylamide gels(26) , and electroblotted onto polyvinylidene fluoride membranes. Membranes were blocked with 5% milk powder in blocking buffer (Tris-buffered saline/0.2% Tween-20) and incubated in 1% milk powder/blocking buffer with polyclonal antibodies, washed extensively with blocking buffer, and then incubated with peroxidase-conjugated anti-rabbit IgG (Jackson Labs). Membranes were washed and visualized using the enhanced chemiluminescence (ECL) method (Amersham Corp.).
The feasibility of the
selection strategy was established by plating TK and
TK
yeast in the presence of growth inhibitory levels
of methotrexate and sulfanilamide in combination with Fig. 1graded concentrations of thymidine (Fig. 1A). TK
yeast failed to grow at
all, whereas TK
yeast grew only in the presence of
sufficiently high concentrations (
1000 µM) of
thymidine that uptake by passive diffusion met cellular requirements
for dTMP. Mammalian NT processes exhibit thymidine affinities in the
low micromolar range (31, 32, 33) .
Therefore, it seemed likely that TK
yeast cells, which
were unable to grow under conditions of thymidylate synthase inhibition
in the presence of low concentrations (
100 µM) of
extracellular thymidine, would be able to grow if they expressed a
functional recombinant NT protein from a cDNA library.
Figure 1:
Identification of a thymidine
transport-specific cDNA by functional complementation of a thymidine
transport defect in S. cerevisiae. A, expression of TK in
yeast allowed salvage of exogeneous thymidine under conditions of dTMP
starvation. TK-expressing (TK) and wild-type
(TK
) KY114 cells were streaked onto media that
contained methotrexate, sulfanilimide, and various concentrations of
thymidine. B, expression of pMTP1 allowed growth of
TK
yeast in the presence of a limiting concentration
of exogeneous dThd. TK
yeast were transformed with
either pYES2 (INSERT
) or pMTP1
(INSERT
) and then tested for growth on medium that
contained methotrexate, sulfanilimide, and either thymidine or
dTMP.
Mouse
leukemia L1210 cells, which possess two distinct equilibrative NT
processes capable of transporting thymidine(8, 34) ,
were used as the source of mRNA for construction of a yeast cDNA
expression library. Yeast were transformed with this library and
assessed for growth in the presence of methotrexate, sulfanilamide, and
100 µM thymidine. A library plasmid, hereafter termed
pMTP1 (plasmid Mouse Transporter Protein 1), was isolated from the
library due to its ability to confer thymidine-dependent growth when
expressed in recipient TK yeast cells (Fig. 1B). Substitution of dTMP for thymidine in the
selection medium failed to rescue pMTP1-expressing yeast, suggesting
that the recombinant protein mediated a selective uptake process rather
than a generalized change in membrane permeability.
Figure 2: MTP-related RNA was expressed in mammalian cells. Preparations of total cellular RNA (20 µg) of mouse L1210/C2, rat Walker 256, human HeLa, and S. cerevisiae KY114 cells were subjected to Northern blot analysis using MTP cDNA as the probe. The arrow indicates the migratory position of mouse 18 S rRNA (1870 nucleotides).
Figure 3:
Primary structure of the MTP cDNA and
protein sequence. A, shown are the nucleotide and predicted
amino acid sequences of MTP cDNA (nucleotides 1-1427) and the
nucleotide sequence of the upstream genomic DNA (nucleotides -610
to -1). The sequence of the synthetic peptide used to produce the
-P antibodies is shown in bold type. There are (i)
consensus phosphorylation sequences for cAMP kinase (residues
7-11), casein kinase II (residues 26-30), and protein
kinase C (residues 10-13) and (ii) two tyrosine-based sorting
motifs (residues 215-218 and 229-232). B,
hydropathic plot of predicted amino acid sequence was produced by the
method of Kyte and Doolittle (37) using a window of 11 amino
acids (MacVector version 4.0).
Assignment of the first ATG
codon of the full-length cDNA as the translational start site of MTP
yielded an open reading frame of 699 nucleotides that encoded a
predicted protein of 233 amino acid residues (26.8 kDa) with four
predicted (37) hydrophobic transmembrane-spanning domains (Fig. 3). The partial cDNA originally isolated appeared to
encode a truncated protein (MTP1) whose translation could have been
initiated at an internal ATG codon (nucleotides 403-405) that
encompassed amino acid residues 90-233 of MTP (16.8 kDa) and only
three of the four transmembrane domains. The predicted open reading
frame of the complete and truncated proteins was confirmed by in
vitro translation of MTP and MTP1 RNA transcripts, which gave Fig. 4rise, respectively, to S-labeled polypeptides
of approximately 24 and 16 kDa (Fig. 4).
Figure 4:
In vitro translation of MTP1 and
MTP RNA transcripts. RNA was produced by in vitro transcription of either MTP1 or MTP cDNA, and rabbit reticulocyte
lysates were then incubated with [S]methionine
in the presence (+RNA) or absence (-RNA) of either MTP1 or
MTP RNA. The translation products were separated by SDS-polyacrylamide
electrophoresis and identified by autoradiography. Shown are migratory
positions of protein molecular mass standards. The arrows indicate translation products.
The translation initiation codon of MTP cDNA and its flanking sequence (5`-TCCGGATGG-3`) did not resemble the ribosome binding consensus sequence (5`-GG(A/G)CCATGG-3`) of higher eukaryotes(21) . In addition, the presumed promoter region of the MTP gene lacked a TATAA transcription initiation sequence motif and contained a sequence motif (CCAAT) that has been implicated in regulation of transcription efficiency(38) . These features are usually associated with ``housekeeping'' genes that are constitutively expressed at low levels in most cells and tissues(39) .
The predicted protein sequence of MTP was found to be 98 and 34% identical to proteins encoded by HUMORF13 and HUMKIAAF (GenBank accession numbers D14696 and D42042, respectively), both of which are human cDNAs of unknown identity and function. The near perfect identity of HUMORF13 suggested that it is the human homolog of MTP.
Figure 5:
Expression of MTP1 and MTP in yeast and Xenopus oocytes. A, yeast were transformed with pYES2
containing either no insert as a control (Cont.), cDNA
encoding the truncated protein (MTP1), or cDNA encoding the
full-length protein (MTP) and grown in galactose-containing
minimal medium to induce expression of the recombinant proteins.
Soluble and insoluble (crude membrane) fractions were isolated and
subjected to SDS-polyacrylamide electrophoresis and immunoblotting.
Replicate samples (25 µg of protein) were probed with anti-fusion
protein or -P antibodies. B, uptake of 100 µM radiolabeled solute (30 min, 20 °C) was measured in Xenopus oocytes that had been injected with water or RNA
produced by in vitro transcription of either MTP1 or MTP cDNA.
The abbreviations used are: dThd, thymidine; Urd,
uridine; Ado, adenosine; Suc, sucrose. C,
crude membrane fractions (25 µg of protein), which were isolated
from oocytes that had been injected with either water or MTP RNA, were
subjected to Western blot analysis with
-P
antibodies.
Functional expression of cDNAs by
microinjection of RNA transcripts into oocytes of X. laevis has recently been used in the isolation and identification of
three different proteins with NT
activity(2, 3, 4) . In this study, Xenopus oocytes were microinjected with RNA transcripts
derived from either MTP1 or MTP cDNA and examined thereafter for their
ability to transport nucleosides. Oocytes injected with MTP1 RNA were
fragile, and many were deformed when compared with either MTP
RNA-injected or water-injected oocytes, necessitating a selection of
apparently ``healthy'' oocytes for use in uptake assays,
which were usually conducted within 2-3 days of RNA injection.
The injection of MTP1 RNA into oocytes stimulated uptake of thymidine,
uridine, and, to a lesser extent, sucrose and had no effect on uptake
of adenosine, relative to water-injected (control) oocytes (Fig. 5B). In contrast, no stimulation of solute uptake
occurred in the oocytes that were injected with MTP RNA (data not
shown) even though membrane-associated recombinant MTP was produced, as
indicated by the reactivity of the -P antibodies (Fig. 5C). Thus, although both the truncated (MTP1) and
complete (MTP) recombinant proteins were produced in the RNA-injected
oocytes, only MTP1 altered membrane permeability.
Figure 6:
A rat homolog of MTP was identified in
Golgi-enriched membranes of rat hepatocytes. Equal quantities (25
µg of protein) of rat liver subcellular membrane fractions were
probed by immunoblotting with -P antibodies. The abbreviations
used are: Total, total cellular protein; Golgi,
Golgi-enriched fraction; Smooth- and Rough-ER, smooth
and rough endoplasmic reticulum fractions; C- and P-Mito, crude and purified mitochondria
fractions.
Figure 7:
A
C-terminal deleted version of MTP-induced thymidine permeability when
produced in Xenopus oocytes. A, schematic
illustration of MTP constructs. Hydrophobic transmembrane domains are
indicated by shading. B, uptake of 100 µM [H]thymidine (30 min, 20 °C) was
measured in oocytes that had been injected with water or RNA
transcripts produced from the various cDNA constructs (MTP1, MTP,
MTP
N, or MTP
C).
The
stimulation of nucleoside uptake observed in Xenopus oocytes
injected with MTPC RNA made it possible to examine the permeant
selectivity of recombinant MTP
C by analysis of inward fluxes of
various [
H] solutes (Fig. 8A) and
inhibition of inward fluxes of [
H]thymidine by Fig. 8competing permeants (Fig. 8B). In these
experiments, MTP
C-expressing oocytes exhibited a 12-fold increase
in uptake of [
H]thymidine and 5-6-fold
increases in uptake of [
H]uridine and
[
H]adenosine relative to water-injected oocytes.
There was no increase in uptake of [
H]sucrose, a
result that differed from that obtained in earlier, similar experiments
with MTP1-expressing oocytes (compare results of Fig. 5B and 8A). Because the uptake of sucrose was negligible in
both water-injected and MTP
C-expressing oocytes, the uptake
induced by recombinant MTP
C appeared to be selective for
nucleosides and not simply the consequence of a change in permeability
of the plasma membrane to low molecular weight solutes.
MTP
C-induced thymidine uptake was mediated because a 100-fold
excess of nonradioactive thymidine completely inhibited uptake of
[
H]thymidine. In addition, the complete and
partial inhibitions of thymidine uptake caused by 100-fold excess
concentrations of dTMP and thymine, respectively, suggested that these
compounds were either inhibitors or substrates of MTP
C.
Figure 8:
MTPC-mediated transport of
nucleosides. A, uptake of 100 µM radiolabeled
solutes by oocytes (30 min, 20 °C) injected with water or MTP
C
RNA. B, uptake of 100 µM [
H]thymidine by oocytes (30 min, 20 °C)
in the absence or the presence of 10 mM nonradioactive
competitive solute. The abbreviations used are: dThd,
thymidine; Urd, uridine; Ado, adenosine; Suc, sucrose; Thy, thymine; dTMP, thymidine
5`-monophosphate.
We have exploited phenotypic complementation of a
pre-existing thymidine transport deficiency in S. cerevisiae to identify membrane proteins that mediate mammalian NT processes.
The complementation strategy required that the recombinant transporter
(i) accepts thymidine as a permeant at concentrations 100
µM and (ii) is targeted to the plasma membrane of yeast.
Although the strategy was devised for isolation of cDNAs encoding NT
proteins of plasma membranes, we describe here its use in the
identification and expression of a cDNA that encodes a previously
unknown protein with nucleoside transport activity that normally
resides in intracellular membranes of mammalian cells.
The protein encoded by the full-length cDNA was provisionally named ``mouse transporter protein'' (MTP). MTP is unusually small (233 amino acids, 26.8 kDa) for a mammalian solute transporter and has only four predicted transmembrane-spanning domains. No homology was found between the amino acid sequences of MTP and any of the plasma membrane NT proteins whose sequences are known(2, 3, 4) . The predicted protein sequence of MTP exhibited high sequence identity (98%) to HUMORF13, an unidentified expressed sequence of human cells (GenBank D14696). Expression of an MTP-related mRNA (presumably encoded by HUMORF13) was demonstrated in this work in a cultured human cell line (HeLa) by Northern blotting.
Our results suggested that MTP is a low abundance protein that may reside in an intracellular membrane compartment rather than the plasma membrane. A protein of the same electrophoretic mobility as MTP was identified in a Golgi membrane-enriched subcellular fraction of rat hepatocytes by Western blotting, although we were unable to visualize MTP by immunofluorescence (data not shown) in either (i) cultured L1210 cells or (ii) in freshly isolated rat hepatocytes. The analysis by immunoblotting of plasma membrane preparations of rat hepatocytes and several cultured cell types (data not shown) revealed no evidence of MTP-reactive material. Finally, although a truncated form of recombinant MTP-mediated transport of thymidine across the plasma membrane of yeast and Xenopus oocytes, full-length recombinant MTP did not. Studies are currently underway to determine the precise subcellular localization of native MTP.
Studies of membrane proteins
that localize to intracellular membrane compartments have revealed a
variety of sorting signals found in either the N- or C-terminal
``cytoplasmic tails'' of the targeted protein (40) .
The importance of residues at the N and C termini of MTP for
subcellular localization was assessed by expressing cDNAs with
terminally deleted sequences (MTPN, MTP
C) in Xenopus oocytes, followed by measurement of induction of NT activity as a
``functional marker'' of recombinant MTP in the plasma
membrane. Thymidine uptake by MTP
C-injected oocytes was increased
>10-fold, whereas thymidine uptake by MTP-injected and
MTP
N-injected oocytes was the same as that observed in the
water-injected oocytes. Because MTP that possessed the C terminus was
directed to an intracellular compartment, a retention signal likely
existed within the 36 amino acids of the C terminus. MTP that lacked
the C terminus was directed, presumably by a default pathway, to the
plasma membrane. The C-terminal region contained two tyrosine-based
sorting motifs (Tyr-X-X-Hydrophobic) previously identified in integral
membrane proteins that are targeted to various compartments in the
Golgi/endosomal/lysosomal pathways(41) . Studies are currently
underway to determine if either (or both) of the tyrosine-based motifs
is required for intracellular localization of MTP. Although the reason
for localization of MTP1 at the plasma membrane of Xenopus oocytes and yeast is unknown, we speculate that the absence of the
first of the four transmembrane domains may have resulted in a
topographical inversion of the C terminus during biosynthesis, thereby
making it inaccessible to the cellular machinary responsible for
retention within intracellular membranes.
The stimulation of uptake
observed in MTPC-expressing oocytes made possible an assessment of
transporter function, although these studies were limited by the
cytotoxic effects of MTP
C expression in oocytes. Uptake of
thymidine was mediated and, because recombinant MTP
C induced
uptake of nucleosides but not of sucrose, appeared to be selective for
nucleosides. MTP
C-mediated transport resembled the well
characterized NT processes of plasma membranes (1) in that both
purine and pyrimidine nucleosides (thymidine, uridine, and adenosine)
were permeants, although the transport characteristics of recombinant
MTP
C expressed in the plasma membranes of Xenopus oocytes
may not faithfully reflect those of the native transporter.
MTP
C-mediate transport was sensitive to inhibition by dTMP and
thymine, suggesting that these compounds may be inhibitors and/or
permeants.
A requirement for intracellular transport of nucleosides is predicted by the compartmentalization of enzymes of nucleoside metabolism within various organelles(41, 42, 43) . Mediated transport of nucleosides has been demonstrated in lysosomes isolated from human fibroblasts(44) , and uptake of nucleosides has been observed in isolated mitochondria(45) . Furthermore, mitochondrial toxicities observed in patients exposed to antiviral nucleosides are thought to require an uptake mechanism for the phosphorylated forms of these nucleoside analogs(46) . The presence of substantial quantities of adenosine has been demonstrated within the lumen of vesicles prepared from fractions enriched for Golgi(47) . However, although Golgi-enriched membranes exhibit mediated exchange of nucleotide-sugars and nucleotides, these processes are not inhibited by the presence of excess nucleosides(48) . In conclusion, MTP is a representative of a new group of transport proteins whose physiologic function may be the transfer of nucleosides between the cytosol and the lumen of intracellular organelles.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34259[GenBank].