©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Membrane Transporter Associated with Intracellular Membranes by Phenotypic Complementation in the Yeast Saccharomyces cerevisiae(*)

(Received for publication, January 4, 1996; and in revised form, February 7, 1996)

Douglas L. Hogue (1)(§) Michael J. Ellison (1)(¶) James D. Young (2)(**) Carol E. Cass (1)(§§)

From the  (1)Departments of Biochemistry and (2)Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (MTPDeltaC) in plasma membranes of Xenopus laevis oocytes. MTPDeltaC-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 MTPDeltaC-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.


INTRODUCTION

Mammalian cells normally possess the capacity for mediated translocation of nucleosides across their plasma membranes (for review see (1) ). These nucleoside-selective transport (NT) (^1)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.


EXPERIMENTAL PROCEDURES

Cell Culture

Mouse leukemia L1210/C2 cells were grown as described previously(8) .

Subcellular Fractionation

Cells were suspended in lysis buffer (0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 mM HEPES, pH 7.5) and Dounce homogenized. The resulting homogenate was centrifuged (1000 times g), and the supernatant was collected and centrifuged (30000 times g), producing nuclei-free membrane (pellet) and soluble cell fractions. Subcellular membrane fractions of rat liver cells were a gift from J. Vance (University of Alberta) and had been prepared and assayed for marker enzmes as described elsewhere(9, 10) .

Xenopus Oocyte Expression Studies

Mature stage IV oocytes were isolated from X. laevis as described(11) . The expression vectors were linearized at a unique restriction site downstream of the cDNA insert and used as template for in vitro synthesis of RNA transcripts using the MEGAscript transcription system (Ambion). Oocytes were microinjected with 20 nl of water or RNA (0.5 mg/ml) and used 2-4 days later for uptake assays (3) or for the isolation of membrane and soluble fractions (12) for use in immunoblotting experiments as described below. Uptake of solute was measured by incubating oocytes (10/assay) at 20 °C for 30 min in transport buffer (100 mM NaCl, 20 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 10 mM HEPES, pH 7.5) that contained a particular radiolabeled solute at a concentration of 100 µM. The oocytes were washed with ice-cold transport buffer, and the radioactivity associated with each oocyte was measured by liquid scintillation counting.

Growth of Yeast

The S. cerevisiae strain KY114 (genotype: MAT, ura3-52, his3-Delta200, trp1-Delta63, ade2-101, lys2-801) was grown in liquid culture or on 1.5% agar plates that consisted of minimal medium containing 2% galactose and appropriate combinations of uracil, tryptophan, histidine, adenine, and lysine as previously specified for plasmid selection(13) . Inhibition of de novo dTMP synthesis was achieved in minimal medium that contained 50 µg/ml methotrexate and 6 mg/ml sulfanilimide (14) .

Generation of TK-expressing Yeast Strain

Molecular biology techniques have been previously described(15) . The Herpes simplex TK gene from pJM81 (14) was amplified by polymerase chain reaction (PCR) using oligonucleotide primers that added EcoRI and SalI sites (5`-GCTAGAATCCGAGTATGGCTTCGTACC-3` and 5`-GCTAGTCGACCGTGTTTCATTAGCCTCC-3`) upstream and downstream of the coding region, respectively. The TK gene was substituted for the ubiquitin gene cassette of the yeast expression plasmid YEp96 (16) such that the start and stop codons of each gene occupied identical positions with respect to the noncoding flanking sequences that comprise the CUP1 promoter and CYC1 transcriptional terminator of YEp96. YEp96 carries the TRP1 auxotrophic selectable marker. The TK-containing plasmid (pDH01) was transformed into KY114 cells, and cells were grown in the absence (constitutive expression) or the presence (induced expression) of 0.1 mM CuSO(4) to assay for TK activity(14) . In all other experiments, TK cells were grown in the absence of CuSO(4).

Production and Screening of the L1210/C2 cDNA Library

Poly(A) RNA was isolated from L1210/C2 cells using the guanidinium thiocyanate/CsCl method (17) and chromatography on oligo(dT)-cellulose. cDNA was prepared using oligo(dT) primer, ligated to BstXI adaptors, and cloned into the pYES2 vector (Invitrogen). pYES2 is a high copy yeast/Escherichia coli shuttle vector that carries the URA3 auxotrophic selectable marker, Gal1 promoter, and CYC1 transcriptional terminator. Following transformation and amplification in E. coli, the cDNA plasmid library (1.2 times 10^6 clones) was recovered and transformed by electroporation (18) into TK yeast, which were plated onto minimal medium that contained 2% glucose in order to repress the Gal1 promoter. The resulting transformants were then transferred onto selection medium (2% galactose-minimal medium plates that contained 125 µg/ml methotrexate, 6 mg/ml sulfanilimide, and 100 µM thymidine). Colonies that arose were streaked onto minimal medium that contained 1 g/liter 5-fluoroorotate and uracil to select for loss of library plasmids. 5-Fluoroorotate-selected isolates were streaked onto fresh selection medium, and isolates exhibiting growth were considered false positives and eliminated from further screening. The URA3-based library plasmids were then isolated from yeast that exhibited both library plasmid- and thymidine-dependent growth on the selection medium.

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.).

Isolation of cDNA by PCR Amplification

The 5` region of the MTP cDNA was obtained using a PCR method. Oligonucleotides that corresponded to regions of the CYC1 terminator of pYES2 (5`-GCGTGAATGTAAGCGTG-3`) and downstream of the termination codon of the MTP cDNA (5`-CTTGCTTAATCTAGATGGTC-3`, XbaI site underlined) were used as primers and the L1210/C2 cDNA plasmid library as template. A PCR fragment that contained an additional 349 nucleotides of 5` cDNA was sequenced and subcloned in-frame (using the internal XbaI site) with the MTP1 cDNA to produce a 1393-nucleotide fragment, termed MTP2. A gt11 mouse brain cDNA library (John Gardner, Fox Chase Cancer Center) was also screened by PCR amplification using gt11 left and right oligonucleotide primers (5`-GGAAGGATCCTGGCGAGGACTCCTGGAGCCCG-3` and 5`-GGAAGTCGACACCAGACCAACTGGTAATG-3`) and an internal MTP-specific oligonucleotide (5`-CCTGACAAAAGCTTTCGGCCC-3`, HindIII site underlined). An additional 34 nucleotides of 5` cDNA was identified and produced as part of a 114-nucleotide SacI-HindIII fragment by PCR amplification using an oligonucleotide that added a 5` SacI site (5`-AGACTGATGAGCTCAGGGCCCGGGCCGAG) and the internal MTP-specific oligonucleotide. This fragment was ligated in-frame into MTP2 cDNA to produce the full-length (1427 nucleotides) MTP cDNA-containing vector (pMTP).

Isolation of the Promoter Region of the MTP Gene

A FIXII mouse genomic DNA library (Stratagene) was screened with P-labeled MTP cDNA probe by hybridization. The resulting positive clones that contained DNA regions that resided 5` upstream of the MTP cDNA were subsequently identified using a P-labeled oligonucleotide (5`-CCTGACAAAAGCTTTCGGCCC-3`) that corresponded to the 5`-untranslated region of MTP cDNA. A HindII genomic DNA fragment (703 nucleotides) was isolated, subcloned, and sequenced.

Construction of Expression Vectors

MTP cDNA was inserted into the mammalian expression vector pcDNA1-AMP (Invitrogen) by first cloning the SacI-MTP-XhoI fragment of pMTP into the pMTL22 vector (20) to produce a unique upstream EcoRI site and then subcloning the EcoRI-MTP-XhoI fragment directly into pcDNA1-AMP. Deletion constructs of MTP were prepared by PCR amplification of MTP cDNA as follows. (i) MTPDeltaN was produced as follows. A construct encoding MTP truncated at its N terminus (lacked amino acid residues 1-24) was produced from pMTP1 using oligonucleotides that corresponded to the CYC1 terminator (5`-GCGTGAATGTAAGCGTG-3`) of pYES2 and an internal region of the MTP cDNA (5`-GGCTCTATAAGCTTGCCACCATGGTCCGCACCGGGACGA-3`, HindIII site underlined). The HindIII-MTPDeltaN-NotI PCR fragment was directly cloned into pcDNA1-AMP. The resulting cDNA encompassed nucleotides 208-1427 of the MTP coding region and contained a Kozak consensus sequence (21) for initiation of translation (5-GCCACCATGG-3`). (ii) MTPDeltaC was produced as follows. A construct encoding MTP truncated at its C terminus (lacked amino acid residues 198-233) was produced using oligonucleotides that corresponded to the T7 promoter region (5`-TAATACGACTCACTATAGGG-3`) of pYES and an internal region of the MTP cDNA (5`-GGCTCTATTCTAGACTATCTCTGGCACATTTCTGTTG-3`, XbaI site underlined). The BamHI-MTPDeltaC-XbaI PCR fragment was cloned into pcDNA1-AMP to yield a coding region that encompassed nucleotides 1-730 of the MTP cDNA and contained a translational stop codon (TAG).

Northern Blots

RNA was electrophoretically resolved on 1.2% agarose-0.67% formaldehyde gels and transferred to nylon membranes. The HindIII-XbaI fragment of MTP cDNA (960 nucleotides) was random primer-labeled with [P]dCTP and incubated with the membranes in aqueous hybridization solution(15) . Blots were washed and then exposed to autoradiographic film.

In Vitro Translation

RNA was prepared by in vitro transcription of MTP1 and MTP cDNAs as described above, and 1-µg quantities were translated in vitro with rabbit reticulocyte lysates (Promega) and TranS-Label (DuPont NEN). Translation reactions were solubilized under reducing conditions, separated on a 12% SDS-polyacrylamide gel, and autoradiographed.

Production of Antibodies and Analysis of Immunoreactivity

Antibodies specific for the N terminus of MTP were generated against a synthetic peptide (TFKRSRSDRFYSTRC) corresponding to amino acid residues 5-19 of MTP that was conjugated to diphtheria toxin (Chiron Mimotopes). Polyclonal antisera against the peptide were raised in rabbits and affinity purified by peptide-Sepharose chromatography(22) ; the resulting anti-peptide (alpha-P) antibodies were shown to discriminate between MTP and MTP1 recombinant proteins. The MTP2 cDNA, which contained the full MTP coding sequence, was cloned in-frame into the pATH11 and pMalcRI vectors (New England Biolabs) to generate trpE-MTP (23) and malE-MTP fusion proteins(24) , respectively. The trpE-MTP and malE-MTP fusion proteins were isolated by electroelution after separation by SDS-polyacrylamide gel electrophoresis. Polyclonal antibodies with cross-reactivity for MTP and MTP1 were generated against the trpE-MTP fusion protein in rabbits, and the antisera were enriched for MTP-specific antibodies by absorption to immobilized malE-MTP fusion protein(25) ; the resulting anti-fusion protein antibodies recognized both MTP and MTP1 recombinant proteins.

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.).


RESULTS

Isolation of a Putative Transporter cDNA by Phenotypic Complementation of an NT Deficiency of S. cerevisiae

S. cerevisiae relies on thymidylate synthase-mediated methylation of dUMP to create dTMP (27) and lacks mechanisms for inward transport of thymidine or dTMP across its plasma membranes(14, 28) . Inhibition of thymidylate synthase in yeast by methotrexate and sulfanilamide leads to depletion of intracellular dTMP and growth arrest(29) . Therefore, simultaneous exposure to these drugs in combination with extracellular thymidine provided a strategy for selection of cells that functionally expressed a mammalian NT protein capable of inward transport of thymidine. In addition to its defect in thymidine transport, yeast lack TK, which is required for conversion of thymidine to a metabolically useful form(30) . To overcome this problem, we created a yeast expression plasmid (pDH01) that contained the H. simplex TK gene under transcriptional regulation of the yeast copper-inducible CUP1 promoter. Introduction of pDH01 into the host strain used for library screening resulted in TK cells that exhibited low levels of constitutive TK activity that could be induced by >10-fold when cells were grown in the presence of 0.1 mM CuSO(4) (data not shown).

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 (geq1000 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 (leq100 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.

MTP1 Encodes a Truncated Version of a Previously Unidentified Protein

The cDNA sequence of pMTP1 encompassed an open reading frame of 451 nucleotides at the 5` terminus, followed by a region of 593 nucleotides that included two sequence motifs (5`-ATTA-3`) previously demonstrated to be signals for selective mRNA degradation(35) , a polyadenylation consensus signal sequence (36) , and a polyadenylated 3` tail. When RNA from S. cerevisiae, mouse L1210/C2 cells (the library source), rat Walker carcinosarcoma cells, and human Hela cervical carcinoma cells were probed with MTP1 cDNA, multiple RNA species of 1400-1500 nucleotides Fig. 2were identified in all of the mammalian RNA preparations (Fig. 2). This suggested that the MTP1 cDNA, which was only 1044 nucleotides, was derived from the 3` region of a larger mRNA that encoded a relatively conserved protein. Additional 5` regions of the mouse MTP cDNA were subsequently isolated from the L1210/C2 cDNA library and a mouse brain gt11 cDNA library using a PCR method and were combined in-frame with MTP1 to yield an apparent full-length cDNA of 1427 nucleotides (Fig. 3).


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 alpha-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.

Production of Truncated and Complete MTP in Yeast and Xenopus Oocytes

MTP was identified because of the ability of a version truncated at the N terminus (MTP1) to complement the thymidine transport deficiency in yeast. However, TK yeast transformed with MTP cDNA failed to grow in the presence of thymidine (data not shown). The failure of MTP cDNA to complement the thymidine transport deficiency was unexpected and suggested that the full-length protein was either absent or functionally defective. To assess these possibilities, polyclonal antibodies were raised against epitopes (i) shared by both MTP and MTP1 or (ii) present in MTP but not in MTP1. The MTP/MTP1 cross-reactive antibodies were raised against a fusion protein (trpE-MTP) and the MTP-specific antibodies (alpha-P) were raised against a synthetic peptide whose sequence was present within the N terminus of MTP (amino acid residues 5-19) and absent from MTP1. These antibodies were used for analysis of extracts prepared from TK yeast that had been transformed with either the full-length (MTP) or the truncated (MTP1) Fig. 5cDNA and grown under nonselective conditions (Fig. 5A). MTP1 cDNA-transformed yeast produced membrane-associated polypeptides that (i) were recognized by the anti-fusion protein antibodies but not by the alpha-P antibodies and (ii) were similar in their electrophoretic mobility to the in vitro MTP1 translation product (compare Fig. 3and 5A). Yeast transformed with full-length MTP cDNA lacked immunoreactive polypeptides altogether, indicating that the failure to complement the transport defect was due to the absence of recombinant protein.


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 alpha-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 alpha-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 alpha-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.

Localization of MTP in Intracellular Membranes of Rat Hepatocytes

The observation that oocytes that expressed membrane-associated MTP did not exhibit altered permeability properties implied that (i) the recombinant protein was present in the plasma membrane but was incapable of mediating solute fluxes across the membrane or (ii) the recombinant protein was functional but present in some other membrane compartment. Native MTP was evidently produced at very low levels, because it could not be detected in L1210/C2 cells (data not shown) by either immunoblotting of membrane preparations or indirect immunofluoresence of cells, even though the cDNA library used to obtain MTP1 cDNA was produced from L1210/C2 mRNA. The presence of MTP-related RNA transcripts in Walker cells (see Fig. 2) suggested the existence of a rat homolog of MTP, and subcellular membrane fractions of rat liver were subjected to Western blot analysis with MTP-specific Fig. 6antibodies (Fig. 6). Immunoreactive material of the mobility (about 24 kDa) expected for mouse MTP was clearly evident in the subcellular fractions that were enriched for Golgi membranes and not in the fractions that were enriched for either endoplasmic reticulum or mitochondrial membranes.


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 alpha-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.



Deletion of the C Terminus Mislocates MTP to the Plasma Membrane

The observation that full-length MTP was found in intracellular membranes indicated that recombinant MTP1, which lacked the N terminus (amino acids 1-83) of MTP, had been mislocated to the plasma membrane in both yeast and oocytes. Because signals for targeting to intracellular membranes have been previously observed in both the N- and C-terminal domains of membrane proteins(40) , the importance of these domains on the subcellular localization of MTP were examined by comparing the effects of deleting the hydrophilic residues at either end of MTP on its ability to stimulate thymidine transport in Xenopus oocytes. RNA transcripts containing the cDNA sequences that encoded the four predicted transmembrane domains of MTP but lacked sequences encoding either 24 residues at the N terminus (MTPDeltaN) or 36 residues at the C Fig. 7terminus (MTPDeltaC) were produced and expressed in Xenopus oocytes (Fig. 7). Thymidine uptake by oocytes injected with MTPDeltaC RNA was >10-fold greater than thymidine uptake by oocytes injected with MTP1 RNA, whereas thymidine uptake by oocytes injected with MTPDeltaN RNA was no different from that of oocytes injected with water (controls) or with MTP RNA. These results implied that the C-terminal domain of MTP likely contained a signal for its retention within intracellular membranes. Many of the MTPDeltaC RNA-injected oocytes displayed unusual morphologies and, as was the case for MTP1 RNA-injected oocytes, only apparently healthy oocytes were used for uptake assays, usually within 2-3 days of injection.


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 [^3H]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, MTPDeltaN, or MTPDeltaC).



The stimulation of nucleoside uptake observed in Xenopus oocytes injected with MTPDeltaC RNA made it possible to examine the permeant selectivity of recombinant MTPDeltaC by analysis of inward fluxes of various [^3H] solutes (Fig. 8A) and inhibition of inward fluxes of [^3H]thymidine by Fig. 8competing permeants (Fig. 8B). In these experiments, MTPDeltaC-expressing oocytes exhibited a 12-fold increase in uptake of [^3H]thymidine and 5-6-fold increases in uptake of [^3H]uridine and [^3H]adenosine relative to water-injected oocytes. There was no increase in uptake of [^3H]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 MTPDeltaC-expressing oocytes, the uptake induced by recombinant MTPDeltaC 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. MTPDeltaC-induced thymidine uptake was mediated because a 100-fold excess of nonradioactive thymidine completely inhibited uptake of [^3H]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 MTPDeltaC.


Figure 8: MTPDeltaC-mediated transport of nucleosides. A, uptake of 100 µM radiolabeled solutes by oocytes (30 min, 20 °C) injected with water or MTPDeltaC RNA. B, uptake of 100 µM [^3H]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.




DISCUSSION

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 leq100 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 (MTPDeltaN, MTPDeltaC) 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 MTPDeltaC-injected oocytes was increased >10-fold, whereas thymidine uptake by MTP-injected and MTPDeltaN-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 MTPDeltaC-expressing oocytes made possible an assessment of transporter function, although these studies were limited by the cytotoxic effects of MTPDeltaC expression in oocytes. Uptake of thymidine was mediated and, because recombinant MTPDeltaC induced uptake of nucleosides but not of sucrose, appeared to be selective for nucleosides. MTPDeltaC-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 MTPDeltaC expressed in the plasma membranes of Xenopus oocytes may not faithfully reflect those of the native transporter. MTPDeltaC-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.


FOOTNOTES

*
This work was supported by the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34259[GenBank].

§
Recipient of a National Cancer Institute of Canada studentship and a student research allowance from the Alberta Heritage Foundation for Medical Research.

Scientist of the Medical Research Council.

**
Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research.

§§
Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. To whom correspondence should be addressed: 356 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 403-492-2139; Fax: 403-492-0886; carol.cass{at}ualberta.ca.

(^1)
The abbreviations used are: NT, nucleoside transport; alpha-P, anti-peptide antibodies; MTP, mouse transporter protein; PCR, polymerase chain reaction; TK, thymidine kinase.


ACKNOWLEDGEMENTS

We thank D. Mowles and S. Yao for technical assistance and J. Vance for providing rat liver membrane fractions.


REFERENCES

  1. Cass, C. E. (1995) in Drug Transport in Antimicrobial and Anticancer Chemotherapy (Georgopapadakou, N. H., ed) pp. 403-451, Marcel Dekker, New York
  2. Pajor, A. M., and Wright, E. M. (1992) J. Biol. Chem. 267, 3557-3560 [Abstract/Free Full Text]
  3. Huang, Q. Q., Yao, S. Y. M., Ritzel, M. W. L., Paterson, A. R. P., Cass, C. E., and Young, J. D. (1994) J. Biol. Chem. 269, 17757-17760 [Abstract/Free Full Text]
  4. Che, M., Ortiz, D. F., and Arias, I. M. (1995) J. Biol. Chem. 270, 13596-13599 [Abstract/Free Full Text]
  5. Hsu, L. C., Chiou, T. J., Chen, L., and Bush, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7441-7445 [Abstract/Free Full Text]
  6. Frommer, W. B., Hummel, S., and Riesmeier, J. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5944-5948 [Abstract]
  7. Riesmeier, J. W., Willmitzer, L., and Frommer, W. B. (1992) EMBO J. 11, 4705-4713 [Abstract]
  8. Hogue, D. L., Hodgson, K. C., and Cass, C. E. (1990) Biochem. Cell Biol. 68, 199-209 [Medline] [Order article via Infotrieve]
  9. Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256 [Abstract/Free Full Text]
  10. Vance, J. E., and Vance, D. E. (1988) J. Biol. Chem. 263, 5898-5909 [Abstract/Free Full Text]
  11. Huang, Q. Q., Harvey, C. M., Paterson, A. R. P., Cass, C. E., and Young, J. D. (1993) J. Biol. Chem. 268, 20613-20619 [Abstract/Free Full Text]
  12. Scotland, P. B., Colledge, M., Melnikova, I., Dai, Z., and Froehner, S. C. (1993) J. Cell Biol. 123, 719-728 [Abstract]
  13. Kaiser, C., Michaelis, S., and Mitchell, A. (1989) Methods in Yeast Genetics , pp. 15-23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. McNeil, J. B., and Friesen, J. D. (1981) Mol. & Gen. Genet. 184, 386-393
  15. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1989) Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York
  16. Ellison, M. J., and Hochstrasser, M. (1991) J. Biol. Chem. 266, 21150-21157 [Abstract/Free Full Text]
  17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  18. Becker, D. M., and Guarente, L. (1989) Methods Enzymol. 194, 182-187
  19. Sanger, F., Niksen, S., and Coulson, A. R. (1977) Proc. Natl. Aced. Sci. U. S. A. 74, 5463-5467
  20. Chambers, S. P., Prior, S. E., Barstow, D. A., and Minton, N. P. (1988) Gene (Amst.) 68, 139-149
  21. Kozak, M. (1991) J. Cell Biol. 115, 887-903 [Abstract]
  22. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , pp. 511-552, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Koerner, T. J., Hill, J. E., Myers, A. M., and Tzagoloff, A. (1991) Methods Enzymol. 194, 477-490 [Medline] [Order article via Infotrieve]
  24. Maina, C. V., Riggs, P. D., Grandea, A. G., Slatko, B. E., Moran, L. S., Tagliamonte, J. A., McReynolds, L. A., and Guan, C. (1988) Gene (Amst.) 74, 365-373
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 18.11-18.18, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  27. Jones, E. W., and Fink, G. R. (1982) The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression , pp. 181-299, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  28. Grenson, M. (1969) Eur. J. Biochem. 11, 249-260 [Medline] [Order article via Infotrieve]
  29. Barclay, B. J., Kunz, B. A., Little, J. G., and Haynes, R. H. (1982) Can. J. Biochem. 60, 172-184 [Medline] [Order article via Infotrieve]
  30. Grivell, A. R., and Jackson, J. F. (1968) J. Gen. Micro. 54, 307-317
  31. Wohlhueter, R. M., Marz., and Plagemann, P. G. W. (1979) Biochim. Biophys. Acta 553, 262-283 [Medline] [Order article via Infotrieve]
  32. Jarvis, S. M., and Griffith, D. A. (1991) Biochem. J. 278, 605-607 [Medline] [Order article via Infotrieve]
  33. Wu, X., Yuan, G., Brett, C. M., Hui, A. C., and Giacomini, K. M. (1992) J. Biol. Chem. 267, 8813-8818 [Abstract/Free Full Text]
  34. Crawford, C. R., Ng, C. Y. C., Noel, L. D., and Belt, J. A. (1990) J. Biol. Chem. 265, 9732-9736 [Abstract/Free Full Text]
  35. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667 [Medline] [Order article via Infotrieve]
  36. Bernstiel, M. L., Busslinger, M., and Strub, K. (1985) Cell 41, 349-459 [Medline] [Order article via Infotrieve]
  37. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  38. Jones, K. A., Kadonaga, J. T., Rosenfeld, P. J., Kelly, T. J., and Tijan, R. (1987) Cell 48, 79-89 [Medline] [Order article via Infotrieve]
  39. Gardiner-Garden, M., and Frommer, M. (1987) J. Mol. Biol. 196, 261-282 [Medline] [Order article via Infotrieve]
  40. Sandoval, I. V., and Bakke, O. (1994) Trends Cell Biol. 4, 292-297 [CrossRef]
  41. Tsiftsoglou, A. S., and Gerogatsos, J. G. (1972) Biochim. Biophys. Acta 262, 239-246 [Medline] [Order article via Infotrieve]
  42. Sano, S., Matsuda, Y., and Nakagawa, H. (1988) J. Biochem. 103, 678-681 [Abstract]
  43. Lindley, E. R., and Pisoni, R. L. (1993) Biochem. J. 290, 457-462 [Medline] [Order article via Infotrieve]
  44. Pisoni, R. L., and Thoene, J. G. (1989) J. Biol. Chem. 264, 4850-4856 [Abstract/Free Full Text]
  45. Walker, L. F., and Lewis, R. A. (1987) Mol. Cell. Biochem. 77, 71-77 [Medline] [Order article via Infotrieve]
  46. Chen, C. H., and Cheng, Y. C. (1992) J. Biol. Chem. 267, 2856-2859 [Abstract/Free Full Text]
  47. Cappaso, J. M., Keenan, T. W., Abeijon, C., and Hirschberg, C. B. (1989) J. Biol. Chem. 264, 5233-5240 [Abstract/Free Full Text]
  48. Hirschberg, C. B., and Snider, M. D. (1987) Annu. Rev. Biochem. 56, 63-87 [CrossRef][Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.