Biochimica et Biophysica Acta 1788 (2009) 2326–2334

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Biochimica et Biophysica Acta

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Disrupted plasma membrane localization and loss of function reveal regions ofhuman equilibrative nucleoside transporter 1 involved in structural integrityand activity

Nicole M.I. Nivillac a, Karanvir Wasal a, Daniela F. Villani a, Zlatina Naydenova a,W.J. Brad Hanna b, Imogen R. Coe a,⁎a Department of Biology, York University, 4700 Keele Street, Toronto Canada M3J 1P3b Department of Biomedical Sciences, University of Guelph, Guelph Canada N1G 2W1

Abbreviations: hENT1, human equilibrative nucleosfluorescent protein; ER, Endoplasmic reticulum; PDI, ProNitrobenzylthioinosine⁎ Corresponding author. Tel.: +1 416 736 2100x3082

E-mail address: coe@yorku.ca (I.R. Coe).

0005-2736/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.bbamem.2009.08.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 April 2009Received in revised form 16 July 2009Accepted 12 August 2009Available online 20 August 2009

Keywords:ENT1Nucleoside transportLocalizationConfocal microscopyProtein foldingStructure

Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transportsnucleosides and analog drugs across cellular membranes. Very little is known about intracellular processingand localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine)result in loss of plasma membrane localization and/or transport function. To understand the role of specificresidues within these regions, we studied the localization patterns of N- or C-terminal deletion and/orsubstitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant andwildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding.Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevisoocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disruptsplasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead tohENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane butshow reduced transport activity. These data suggest that these two regions contribute to the structuralintegrity and thus correct processing and function of hENT1.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Nucleoside transporters (NTs) play an essential role in thetransport of nucleosides and nucleoside analog drugs, such asgemcitabine and fialuridine, across cellular membranes [1–3]. NTshave been classified into two families known as concentrative (C) NTsand equilibrative (E) NTs. CNTs use ion (i.e. Na+) gradients to co-transport nucleosides across the membrane, while ENTs transportnucleosides in a passive manner down an endogenous concentrationgradient [4,5]. ENTs are ubiquitously expressed, in contrast to theCNTs, which have a more limited tissue distribution [6,7].

ENTs belong to the SLC29 transmembrane protein family ofnucleoside transporters [4]. The predicted topology of ENTs suggeststhat these proteins have a unique 11 transmembrane structure withan intracellular N-terminus and an extracellular C-terminus [8,9]. Todate, four isoforms of these proteins have been identified, ENT1–ENT4

ide transporter 1; GFP, Greentein disulfide isomerase; NBTI,

5; fax: +1 416 736 5698.

ll rights reserved.

[4,10]. Among the best characterized ENTs are the human ENTs, orhENTs, particularly hENT1 [4]. The hENT1 protein is primarilylocalized to the plasma membrane although [1] have proposed thathENT1 is also targeted to the mitochondria [1,11] following ER export.However, the regulatory signals and mechanisms underlying intra-cellular processing, targeting/trafficking or function for all ENTs arepoorly understood.

In order for membrane proteins, such as hENT1, to localize to theplasma membrane, they must undergo correct processing, targetingand trafficking. These processes are aided by many differentinteracting partners that regulate folding, ER export and/or incorpo-ration into vesicles [12]. Correct folding ofmembrane proteins is aidedby chaperone proteins such as binding protein (BiP), which associateswith hydrophobic residues and, in an ATP dependent manner,“guides” the alignment of peptides that will later be linked bydisulfide bridges formed by protein disulfide isomerase (PDI) [12–16].Besides folding, interacting proteins also play a role in allowing aprotein to travel along the secretory pathway to its final destination.These interacting partners (i.e. coatomer proteins or clathrin) allow aprotein to be incorporated into vesicles for transport and laterincorporation into their target membranes. For all these packaging/translocation steps, specific motifs within the protein sequence are

Table 1Primers used in the construction of the truncation and substitution mutants of GFP-hENT1 The C-terminal amino acid sequences of wildtype GFP-hENT1 and the variousmutants are shown along with the primers (in 5′ to 3′ direction) used to generate them.

Primer Primer sequence Mutant

hENT1Fwd

CGG GGT ACC CCG ATG CGG CTA AAC ATG ACA ACC AGTCAC CAG CCT CAG

N/A

hENT1-T2reverse

TCC CCG CGG GGA TCA TGC CTC GAA CAG GAA GGA GAAAAC AGC CCC CAG TGC

SFLFRA - -

hENT1-T4reverse

TCC CCG CGG GGA TCA GAA TCA GAA CAG GAA GGA GAAAAC AGC CCC CAG TGC CAG ACC

SFLF - - - -

hENT1-T6reverse

TCC CCG CGG GGA TCA GAA GGA GAA AAC AGC CCC CAGTGC CAG ACC CAG ACA

SF - - - - - -

hENT1-Areverse

CCG CGG GGA TCA CAC AAT TGC CCG TTT CAG GAA GGAGAA AAC AGC CCC

SFLKRAIV

hENT1-Creverse

CCG CGG GGA TCA CAC AAT TGC CCG TTT CTT TTT GGAGAA AAC AGC CCC

SKKKRAIV

hENT1-Dreverse

TCC CCG CGG GGA TCA CAC AAT TGC CCG GAA CAG TTTGGA GAA AAC AGC CCC

SKLFRAIV

hENT1-Ereverse

TCC CCG CGG GGA TCA TGC CTC GAA CAG GAA GGA GAAAAC AGC CCC CAG TGC

SFLFEAIV

hENT1-Greverse

TCC CCG CGG GGA TCA CAC AAT TGC CCG TGC AGC TGCGGA GAA AAC AGC CCC

SAAARAIV

hENT1-Hreverse

TCC CCG CGG GGA TCA CAC AAT TGC CCG GAA CAG GGCGGA GAA AAC AGC CCC

SALFRAIV

hENT1-Ireverse

TCC CCG CGG GGA TCA CAC AAT TGC CCG GGC CAG GAAGGA GAA AAC AGC CCC

SFLARAIV

hENT1-Jreverse

TCC CCG CGG GGA TCA CAC AAT TGC GAG GAA CAG GAAGGA GAA AAC AGC CCC

SFLFLAIV

hENT1-Kreverse

TCC CCG CGG GGA TCA CAC AAT TGC GTG GAA CAG GAAGGA GAA AAC AGC CCC

SFLFHAIV

hENT1-Lreverse

TCC CCG CGG GGA TCA CAC TTT TGC CCG GAA CAG GAAGGA GAA AAC AGC CCC

SFLFRAKV

The specific truncation or residue substitutions are indicated in bold.

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required [17]. The presence of specific amino acid motifs, in a correctstructural conformation, is therefore crucial for precise proteinfolding, ER export and trafficking and single point mutations in aprotein sequence can significantly affect any one of these steps [18].

In recent years, many studies have shown that the N- and/ or C-termini have a regulatory role for a large number of membraneproteins. For instance, the human kidney anion exchanger (kAE1)possesses two tyrosines (one located at the N-terminus and onelocated at the C-terminus) which play a role in correct basolateraltrafficking of kAE1. Disruptions to these two residues result inretention of the protein within the trans-Golgi network [19].Similarly, alanine substitutions in the C-terminus of the multidrugresistance P-glycoprotein ABC-type transporter (P-gp), result inmisfolding and arrested cell surface expression [20] and a numberof other studies on the processing of transporters (e.g. DAT, hSVCT1,GAT1) have demonstrated that motifs involved in ER export and/orplasma membrane targeting are located in the C-terminus [21–24].Disruption of thesemotifs results in retentionwithin the ER, likely dueto ER quality control mechanisms [25]. In addition, studies (e.g. on theCystic fibrosis transmembrane conductance regulator) show that theC-terminus contributes to protein function through interaction withother proteins [26]. These data suggest that the C-terminus of atransporter (which is typically intracellular in these studies) is acommon location for regulatory motifs. However, the predictedstructure of hENT1 possesses an extracellular C-terminus callinginto question the role of this region of the protein for this transporter.Indeed, virtually nothing is known about folding, ER processing ortrafficking of hENT1. A small number of individual residues in ENT1(or orthologs) have been implicated in targeting and function in otherstudies [i.e. 27–29]. However, the existence of regulatory motifs andthe underlying mechanisms involved in regulation of hENT1 proces-sing and localization are still largely unknown.

To better understand processing and localization of hENT1, westudied the contribution of the C-terminus, a region implicated intargeting in other transporters [e.g. 30]. We also looked at thecontribution of the very highly conserved PWN motif at the N-terminus. This motif is present in almost every member of the ENTfamily identified to date and is therefore likely to have a critical role inhENT1 biology [10].

2. Materials and methods

2.1. Generation of the GFP-hENT1 mutants

Mutations to the PWN triplet were introduced using theStratagene Quikchange mutagenesis kit (Statagene, CA, USA) onto aGFP-hENT1 template following the manufacturer's instructions.

Primers for the C-terminal mutants were synthesized whichintroduced a KpnI restriction site before the GFP-hENT1 start codon,and a stop codon followed by SacII site after the last eight amino acidsof the hENT1 C-terminus. (Table 1 lists all primers). A unique NotIrestriction site was introduced between the KpnI site and the startcodon for identification of positive clones after ligation. None of theintroduced restriction sites altered the protein sequence. All themutants were then amplified by means of Polymerase Chain Reaction(PCR) using GFP-hENT1 as a template.

A KpnI/SacII vector fragment from pEGFP-C1 (Clontech Laborato-ries Inc., Mississauga, ON, Canada) and a KpnI/SacII GFP-hENT1 insertfragment were isolated using the Gel Extraction Kit (Qiagen Inc.,Canada) and ligated using T4 DNA ligase (New England BioLabs;Pickering, ON, Canada). Ligation reactions were incubated overnightat 16 °C and then transformed into Novablue competent cells (EMDBiosciences, Mississauga ON, Canada). Aliquots were plated on LB-agar supplemented with 50 μg/mL kanamycin. Single colonies fromthese plates were grown in LB medium supplemented withkanamycin. Plasmid DNA was isolated from bacterial cultures using

the Genelute HP Plasmid Mini Prep Kit (Sigma-Aldrich, Oakville ON,Canada) and analyzed on a 1% (w/v) agarose gel following either aNotI digestion or a double digestion of SacII and NotI (New EnglandBioLabs; Pickering, ON, Canada).

Positive clones for the PWN and C-terminal mutants weresequenced in both forward and reverse directions at the YorkUniversity Core Molecular Sequencing Facility. Samples showing thecorrect sequence, including the desired mutation (if applicable) werepurified using the Endo-Free Maxi prep Kit (Qiagen Inc., Canada).

2.2. Cell culture

COS-7 and MCF-7 cells were maintained in Dulbecco's ModifiedEagle's Medium (DMEM; Gibco BRL, MD, USA) supplemented with10% (v/v) fetal bovine serum (FBS; Gibco BRL, MD, USA). Cells weregrown on 10 cm2 plates at 37 °C in a humidified incubator and a 5%CO2 atmosphere.

2.3. Cell transfection for confocal analysis

COS-7 and MCF-7 cells were seeded in 6-well plates on glasscoverslips and allowed to grow at 37 °C in 2 mL DMEMwith 10% (v/v)FBS for 24h to a density of approximately 80%. One hour prior totransfection, the medium was replaced with 1 mL fresh DMEM with10% (v/v) FBS. For the transfections, 2 μg of plasmid DNA was mixedwith 250 μl DMEM (without FBS). In a separate vial, 12 μl ofLipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA) was added to100 μl FBS free DMEM. The two solutions were incubated separatelyfor 5min then mixed together and incubated at room temperature for20min. The mixture was then added drop wise to cells. The 6-wellplateswere incubated overnight in a humidified incubator at 37 °C and5% CO2 atmosphere. The transfected cells were fixed with 2% (w/v)paraformaldehyde (PFA) 20h post transfection and subjected toorganelle labeling.

Table 2Localization of the hENT1 PWN mutants (residues 28–30).

Mutant MutantSequence

Localization

PM I

Wt hENT1 PWN +++ +PWA PWA +++ +PAN PAN +++ +PAA PAA +++ +AWN AWN − +++AWA AWA − +++AAN AAN − +++AAA AAA − +++pEGFP-C1 – − +++ (diffuse)

Substitutions are indicated in bold and are underlined.PM plasma membrane.I Intracellular.+++ ≥60 of cells showing hENT1 localization in indicated area.++ 40–59% of cells showing hENT1 localization in indicated area.+ 6–39% of cells showing hENT1 localization in indicated area.− ≤5% of cells showing hENT1 localization.

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2.4. Cell transfection for NBTI binding assay

COS-7 or MCF-7 cells were seeded in 10 cm2 plates and allowed togrow overnight at 37 °C in 10 mL DMEMwith 10% (v/v) FBS to a densityof approximately 80%. One hour prior to transfections, the medium inall plates was replaced with 4 mL fresh DMEM with 10% (v/v) FBS.For transfections, 15 μg of plasmid DNA was mixed with 1.5 mL DMEM(without FBS). In a separate vial, 80 μl of LipofectAMINE 2000(Invitrogen, Carlsbad, CA, USA) was added to 1.5 mL DMEM withoutFBS. The two solutions were incubated as described above and the finalmixturewas slowlypouredonto cells. Plateswere incubatedasdescribedabove. Cells were collected 18h post transfection. Equivalency of trans-fection across the experiments was determined by comparing the totalpercentage of fluorescing cells between transfected samples within asingle experiment. Experiments with less than 65% transfection effi-ciency were not used. GFP-hENT1 wildtype, which shows characteristicplasmamembrane staining, was used as a positive control while pEGFP-C1emptyvector,which showsdiffuse cytoplasmic staining,wasusedas anegative control (see Fig. 1A and B respectively) in all experiments.

2.5. Labeling of organelles and immunocytochemistry

Transfected cells were treated with the intracellular dyes Lyso-Tracker Red DND-99 or MitoTracker Red CMXRos (Molecular ProbesInc. Eugene, OR) for 30min to label the lysosomes or mitochondriarespectively. Labeled cells were then mounted cell side down on glassmicroscope slides using one drop of mounting medium (DAKOcyto-mation, Mississauga, ON, Canada).

Immunocytochemistry was performed using the SelectFX™ AlexaFluor® 488 Endoplasmic Reticulum Labeling Kit (Molecular Probes,

Fig. 1. Confocal microscopy of mammalian cells transfected with PWN GFP-hENT1mutants. A. pEGFP-hENT1 wildtype; B. GFP-empty vector negative control; C. hENT1-PAN; D. hENT1-PWA; E. hENT1-PAA; F. hENT1-AAN; G. hENT1-AWN; H. hENT1-AAA.Presence of the proline residue results in increased GFP-hENT1 at the plasmamembrane compared to mutants lacking the proline. Bar=10 μm.

Invitrogen, Carlsbad, CA, USA) forfixed cells. The sampleswere treatedaccording to the manufacturer's protocol except that instead of usingthe AlexaFluor® 488 goat anti-mouse IgG as a secondary antibody,AlexaFluor® 594 goat anti-mouse IgG was used since the 488 variantshows a green signal which would be indistinguishable from the GFPsignal. Coverslips were mounted cell side down as described above.

All slides were viewed at the York University Microscopy andImaging Facility using the Olympus Fluoview 300 confocal microscope(60× oil immersion lens, 1.4 N.A.) and Olympus Fluoview 300 imagingsoftware (version 4.3). A minimum of 10 fields of view were analyzedto determine localization patterns for pEGFP-C1 empty vector, hENT1wildtype and each mutant.

All the confocal images show a single, representative, section of aZ-series taken through the entire cell. The section was chosen to showplasma membrane localization or ER localization.

ToquantifyhENT1presenceat theplasmamembraneon confocal, thepercentage of cells showing plasma membrane localization was deter-mined and compared to thenumberof cells showing intracellular hENT1,for an average of 10 fields of view. Observations were done withoutknowledge of themutant being viewed. Cells showing definitive plasmamembrane staining withminimal internal localization were classified asthe protein being present at the plasma membrane. Cells lacking defini-tive membrane staining were classified as the protein being retained.

A complete list of all the mutants and their localizations is shownin Tables 2–5.

Table 3Localization and function of the PWN mutants.

Sample % cells showing plasmamembrane fluorescence

% NBTI bindingversus GFP-hENT1

% function versusGFP-hENT1

PWN 71±2.2 100±0 100±0pEGFP-C1 0±0 6±0.6 0±0PWA 55±3.1 76±17.6 37.2±16.1PAN 59±1.5 74±13.1 29.8±13.3PAA 31±3.5 49±10.1 26.8±18.5AWN 6±1.2 NT 3.5±1.5AWA 1±0.3 NT NTAAN 2±0 25±5.5 NTAAA 0±0 NT NT

The percentage of cells where fluorescence was observed at the plasma membrane isbased on analysis of 10 fields of view in at least three independent experiments (n≥3).Data are presented as an average±S.E. NBTI binding of each sample is represented aspooled data±S.E, from at least two independent experiments (n≥2), each bindingassay conducted in duplicate. Values are presented as an average compared to NBTIbinding observed with wildtype GFP-hENT1, which is set at 100%. Function (transportactivity) is presented as pooled data±S.E, from at least three independent experiments(n≥3), with approximately 15 oocytes used per assay. Values are presented as anaverage compared to wildtype GFP-hENT1, which is set at 100% activity.NT not tested.

Table 4Localization of the hENT1 C-terminal mutants.

Mutant Mutant sequence Mutation type Localization

PM I

wt hENT1 SFLFRAIV – +++ +hENT1-T2 SFLFRA - - Truncation + +++hENT1-T4 SFLF - - - - Truncation + +++hENT1-T6 SF - - - - - - Truncation − +++hENT1-T8 - - - - - - - - Truncation − +++hENT1-A SFLKRAIV Non-polar → positive + +++hENT1-C SKKKRAIV Non-polar → positive − +++hENT1-D SKLFRAIV Non-polar → positive + +++hENT1-E SFLFEAIV Positive → negative ++ ++hENT1-G SAAARAIV Non-polar → non-polar ++ ++hENT1-H SALFRAIV Non-polar → non-polar +++ +hENT1-I SFLARAIV Non-polar → non-polar +++ +hENT1-J SFLFLAIV Positive → non-polar +++ +hENT1-K SFLFHAIV Positive long chain → positive bulky chain +++ +hENT1-L SFLFRAKV Non-polar → positive + +++hENT1-long SFLFRAIVGAARDPPDLDN Extension − +++pEGFP-C1 – – − +++ (diffuse)

Truncation or residue substitutions are indicated in bold and are underlined.PM plasma membrane.I intracellular.+++ ≥60 of cells showing hENT1 localization in indicated area.++ 40–59% of cells showing hENT1 localization in indicated area.+ 6–39% of cells showing hENT1 localization in indicated area.− ≤5% of cells showing hENT1 localization in indicated area.

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2.6. Nitrobenzylthioinosine (NBTI) binding assays

Nitrobenzylthioinosine (NBTI) is a tight binding, high affinity(nM), specific inhibitor of hENT1 [8]. Measurement of levels of freeversus bound NBTI in whole cells is frequently used as an indication ofthe amount of ENT1 present at the plasma membrane [31]. Confocaldata were therefore complemented by NBTI binding assays whichprovide an additional quantitative analysis of hENT1 protein at themembrane. NBTI binding assays were done using standard procedures[32] at room temperature in a 1 mL final volume of NBTI bindingbuffer (10 mM Tris–HCl, 100 mM KCl, 0.1 mM MgCl2•6H2O, 0.1 mMCaCl2•2H2O at a final pH of 7.4). Samples were incubated in the

Table 5Determination of C-terminal mutant localization and function.

Sample Sequence % cells showingplasmamembranefluorescence

% NBTI bindingversusGFP-hENT1

% functionversusGFP-hENT1

hENT1 wildtype SFLFRAIV 71±2.2 100±0 100±0pEGFP-C1 – 0±0 6.0±0.7 0±0hENT1-T2 SFLFRA - - 27±1.5 14±8.5 0±0hENT1-T4 SFLF - - - - 16±2.0 19±1 0±0hENT1-T6 SF - - - - - - 0±0 NT NThENT1-T8 - - - - - - - - 0±0 8±1.5 2.8±1.0hENT1-A SFLKRAIV 13±3.3 NT 2.5±2.5hENT1-C SKKKRAIV 0±0 3±2.5 NThENT1-D SKLFRAIV 41±10.5 48±2.6 0.5±0.5hENT1-E SFLFEAIV 51±10.0 56±11.0 45.8±11.5hENT1-G SAAARAIV NT 51±9.0 31.6±14.5hENT1-H SALFRAIV 64±3.5 87±13.5 54.6±13.8hENT1-I SFLARAIV 68±1 70±13.3 43.25±4.8hENT1-J SFLFLAIV NT 83±16 40.9±10.5hENT1-K SFLFHAIV 69±0.5 81±19.0 58.8±5.71hENT1-L SFLFRAKV NT 29±7.5 36.17±10.9

The percentage of cells where fluorescence was observed at the plasma membrane isbased on an average of 10 fields of view in at least three separate trials. Data arepresented as an average±S.E. NBTI binding is tested in duplicate within each assay andvalues are represented as an average percentage versus wildtype hENT1, with wildtypehENT1 considered to be 100%. NBTI binding of each sample is represented as pooleddata from n≥2±S.E. Function is presented as pooled data±S.E. from 15 oocytes persample per trial, with at least three trials for control or mutant.NT not tested.

presence or absence of 10 μM NBTI with [3H] NBTI ranging inconcentration from 0.2nM to 7.5nM (Moravek, CA, USA) [32]. Foreach analysis, pEGFP-C1 was used as a negative control (with NBTIbinding levels reflecting endogenous levels of ENT1 in both COS-7 andMCF-7 cells) and pEGFP-hENT1 was used as a positive control (withNBTI binding levels reflecting the degree of over-expression of GFP-hENT1 in both COS-7 and MCF-7 cells). All samples were incubatedfor 50min after addition of 100 μl transfected cell suspension(~1.5×105 cells). Reactions were stopped with 5 mL of ice coldbinding buffer. The solutions were then filtered through WhatmanGF/B filters (Fisher Scientific, Ottawa, Canada) and washed with anadditional 8 mL of binding buffer. Radioactivity levels were measuredusing a scintillation counter (1600 TR Scintillation Analyzer, Packard)and then subjected to non-linear regression analysis (GraphPad,PRISM version 4).

2.7. cRNA synthesis

To allow for in vitro transcription, a T7 promoter sequence wasintroduced before the start codon of each mutant as indicated in themMessage mMachine kit (Ambion, Texas, USA) instruction manual.PCR products were sequenced as previously described. RNA tran-scripts were subsequently generated according to the mMessagemMachine kit manufacturer instructions. The concentration and sizeof the RNA transcripts was determined using a spectrophotometerand agarose gel electrophoresis.

2.8. Oocyte extraction and injection

To test functionality Xenopus laevis oocytes were used forheterologous expression since they lack endogenous nucleosidetransport activity [31]. Oocytes were removed from adult femalefrogs as described previously [34]. After extraction, stages V–VIoocytes were stored overnight at 18–20°C in ND96 medium pH7.6[96 mM NaCl, 2 mM KCl and 5 mM Hepes supplemented withgentamicin sulphate (50µg/ml), penicillin G (100µg/ml), 2 mMpyruvic acid, 90 mg theophylline, 1 mM MgCl2 and 1.8 mM CaCl2].Follicle layers were removed by treating the oocytes with collagenaseType I (1 mg/ml) (Sigma-Aldrich, Oakville ON, Canada) for 20–40minin calcium-free saline.

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For injections, 18 ng of mutant hENT1 cRNA, wildtype hENT1(positive control) or water (negative control) was individuallyinjected into ~15 oocytes per treatment (mutant, positive control orwater) per trial using a nanoinjector (Drummond Scientific Company,PA, USA). Following injection, oocytes were kept at room temperaturefor 72h in ND96++++ medium. To assess transport activity, oocyteswere first incubated for 15min in choline chloride transport buffer(100 mM ChCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mMHepes, at pH7.5). The transport buffer was then removed and oocyteswere incubated for 60min in 200 µl fresh choline chloride transportbuffer containing 100 µM uridine and 0.1 mCi/ml [3H] uridine.Following incubation, extracellular uridine was removed by sixrapid 1 mL washes (all carried out within one minute) using NBTIcontaining choline chloride transport buffer (NBTI prevents nucleo-side efflux during the washes). Each single oocyte was then placedinto a separate scintillation vial containing 200 µl of 1% (w/v) SDS.Vials were subjected to vigorous shaking for 45min to dissolve theoocyte. Finally, 2 ml of scintillation fluid was added to each vialfollowed by liquid scintillation counting using the 1600 TR Scintilla-tion Analyzer (Packard).

3. Results

3.1. Analysis of location of wildtype hENT1 confirms predictedlocalization patterns

The use of recombinant tagged-hENT1 is well established as amethod for the study of hENT1 [e.g. 35] and many studies showthat hENT1 is typically localized to the plasma membrane [29,31].GFP-tagged hENT1 was used to study localization patterns since noreliable commercial antibody against endogenous hENT1 exists forconfocal microscopy. Transfection of GFP-hENT1 into both MCF-7 andCOS-7 cells showed predominantly membrane localization patterns asexpected (Figs. 1A and 2A), suggesting that no difference in expression/localization exists between these two cell types.Wedid not observe anyGFP-hENT1 co-localization with the mitochondria (as has been sug-gested in previous studies) [1]. Localization patterns of all substitutionand deletion mutants were compared to GFP-hENT1 wildtype. Basedon confocal analyses, approximately 70% of wildtype GFP-hENT1 waspresent at the plasma membrane. The remaining GFP-hENT1 is foundintracellularly where it is most likely undergoing processing within thesecretory pathway. pEGFP-C1, the GFP-empty vector, was used as anegative control and showed no plasma membrane localization butrather, a diffuse cytoplasmic staining (Figs. 1B and 2B).

3.2. Presence of P28 within the N-terminal PWN triplet is required forplasma membrane localization

The PWN motif is found in almost every ENT family memberidentified to date (Fig. 3A) [10].We therefore determinedwhether theresidues whichmake up the PWN sequence play a role in the ability ofhENT1 to reach the plasma membrane by substituting one or moreresidues within this triplet with alanines. Our data showed thatpreservation of P28 within the PWN triplet led to the presence ofmore GFP-hENT1 at the plasma membrane compared to mutantslacking P28 (Fig. 1, Tables 2 and 3). Replacing either W29 or N30 withan alanine (while preserving P28) reduced localization at the plasmamembrane by approximately half based on confocal microscopy;Table 3. However, replacing both W29 and N30 with alanines(without mutating P28) resulted in about one third of the controllevel of GFP-hENT1 expression at the plasma membrane suggestingthat the presence of either residue, in combination with P28, isnecessary for effective plasma membrane localization (Table 3). GFP-hENT1 mutants, where P28 was substituted with an alanine, showvery low levels of plasma membrane localization regardless of thepresence of other components of the triplet.

Since ER quality control mechanisms can cause retention ofproteins [15] we determined if the mutants were localized to the ERby determining if they were co-localizing with a well established ERmarker, protein disulfide isomerase (PDI). Some ER co-localization ofthe PWN mutants was observed suggesting that mutations in thePWN motif led to possible misfolding or ER export motif disruption.However, intracellular localization was not exclusive to the ER butalso to other unidentified structures.

3.3. Truncation of two, four, six or eight C-terminal amino acids result inloss of plasma membrane localization

We had initially found that elongating the C-terminus by 11 aminoacids resulted in almost complete abolition of cell surface expressionsuggesting that the integrity of the C-terminus was important inplasma membrane localization. Therefore, to explore the possiblerole of the C-terminus in directing or regulating localization of hENT1to the plasma membrane, we made various truncation mutants(Table 4). We observed that removal of the last two amino acids (ΔIV,mutant T2) or last four amino acids (mutant T4, ΔRAIV) resulted inthe majority (~75% or more) of GFP-hENT1 proteins being retainedwithin the cell, (Table 5). Removal of the last six (mutant T6,ΔLFRAIV)or eight amino acids (mutant T8, ΔSFLFRAIV) resulted in no plasmamembrane localization. We hypothesized that mutant hENT1 wasbeing retained within the ER and confirmed ER co-localization usingthe PDI antibody described above. Our results show that wildtypeGFP-hENT1 is primarily localized at the plasma membrane (Fig. 2A),while the T6 and T8 mutants both show retention of the mutantwithin the cell including co-localization with the ER (e.g. mutant T6,Fig. 2C). Mutants T2 and T4 show both plasma membrane and ER co-localization (i.e. mutant T2 Fig. 2D and E). Taken together, these datasuggest that the last 8 amino acids may be critical for folding/ERexport and plasma membrane localization. Therefore, we nextdetermined which specific residues were responsible for these effects.

3.4. Identification of specific C-terminal residues involved in correctlocalization of hENT1

While truncation mutations suggest that an intact hENT1 C-terminal region is required for localization to the plasmamembrane, itis not clear whether specific residues or motifs within this region arerequired. Therefore, substitutions were introduced into the SFLFRAIVregion of the hENT1 C-terminus and localization patterns wereanalyzed by confocal microscopy (Table 4, mutants A–L). Amino acidsubstitutions preserve the length of the protein and thus effects aredue to changes in residue rather than a truncated protein as has beendemonstrated in similar studies with other transporters [e.g. 23,24].

Since the highly hydrophobic triplet, FLF, is conserved in bothhENT1 and hENT2, which both localize to the plasma membrane[6,36] as well as a many (but not all) other ENTs (Fig. 3B), wehypothesized that this might be an important sequence for allowinghENT1 to reach the plasma membrane. We determined the effect ofcharge reversal within this triplet by introducing positively chargedlysines (K, mutants A–D; Tables 4 and 5). In addition, non-polaralanines were substituted to study the effect of changing the specificresidues in the FLF motif but conserving the charge (mutants G–I). Acharge substitution mutant was also made by introducing a lysinein the second hydrophobic triplet present in the C-terminal, AIV(mutant L; Table 4).

Substitution of a single positive charge in the FLF triplet,substantially reduces the overall presence of hENT1 at the plasmamembrane (mutants A and D; Table 5) while mutation of all threeresidues of the FLF amino acid sequence (mutants C; Table 5) resultsin a complete loss of hENT1 expression at the plasma membrane withconcomitant ER co-localization visible suggesting retention of themutated hENT1 in the ER (Fig. 2F, mutant C, FLF to KKK).

Fig. 2. Confocal analysis of GFP-hENT1 C-terminal truncation and substitution mutants in COS-7 cells. A. GFP-hENT1 wildtype. B. pEGFP-C1 empty vector. C. GFP-hENT1-T6colocalizes with the ER. D. GFP-hENT1-T2 is present at the plasmamembrane. E. GFP-hENT1-T2 colocalizes with the ER (yellow inmerged image). F. GFP-hENT1-Cmutant colocalizeswith the ER and unidentified intracellular structures. G. GFP-hENT1-E is present at the plasma membrane. H. GFP-hENT1-E colocalizes with the ER. PDI=protein disulfide isomeraseER marker. Bar=10 μm.

2331N.M.I. Nivillac et al. / Biochimica et Biophysica Acta 1788 (2009) 2326–2334

Fig. 3. ClustalW alignment of ENT orthologs showing the highly conserved regions ofstudy. A. Selection of ENTs from diverse taxons showing conservation of the PWNmotif(boxed). B. Selection of ENTs from diverse taxons showing C-terminus where thehydrophobic triplet–charged residue–hydrophobic triplet pattern is present at least inpart. Asterisks represent identical residues, colons represent similar residues.

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Our data also show that single alanine substitutions within the FLFtriplet, which do not reverse the charge but rather conserve thehydrophobicity, have very little effect on the localization of the proteinsince it is found predominantly at the plasma membrane (mutants Handmutant I; Table 5).However,when all three residues are replaced byalanines, the mutant hENT1 is predominantly retained (mutant G;Table 5) compared to wildtype hENT1. Introduction of a lysine into thesecondhydrophobic triplet in theC-terminus (AIV), leads toa significantretention of hENT1 (mutant L; Table 5). This suggests that conservationof the hydrophobic character of the residue at this site is important forhENT1 localization.

To investigate the role of the positively charged arginine (R) inlocalization, a negatively charged glutamic acid (E) or a non-polarleucine (L) was substituted (Table 4). In addition, since argininepossesses a long side chain, we also determined whether substitutionwith another positive amino acid containing a bulky side chain,histidine (H), would affect plasma membrane localization.

Results show that the effect of the substitution is dependent onthe charge of the substituted residue. Thus, introduction of a negativeglutamic acid (E) results in less protein at the plasma membranecombined with evidence of ER co-localization, (Fig. 2G and H).However, introduction of a non-polar leucine (L) or a bulky positivehistidine (H) has no effect on plasma membrane localization(Table 5). These data suggest that either a positive or non-polarresidue at the−4 position of the C-terminus is required for hENT1 tobe present at the plasma membrane and that a negatively chargedresidue in this position disrupts processing and therefore plasmamembrane localization.

3.5. Determination of hENT1 presence at the plasma membrane by NBTIbinding confirms confocal results

To confirm our plasma membrane localization data derived fromconfocal analyses, we conducted binding assays for selected mutants.

Results demonstrate that transfection of wildtype GFP-hENT1 intoCOS-7 cells shows the highest level of NBTI binding (defined as 100%)demonstrating high levels of plasmamembrane expression comparedto the negative control which showed 10% NBTI binding relative to theoverexpressed GFP-hENT1 which is consistent with NBTI binding byendogenous ENT1 in these cells (Table 5).

The requirement for the presence of P28 within the PWN triplet ineffective plasma membrane localization was confirmed in thatsubstitution of either the tryptophan (W) or the asparagine (N) inthe triplet did not significantly affect binding levels compared tocontrol (Table 3). However, mutation of both W29 and N30simultaneously strongly reduced binding suggesting that W29 and/or N30 need to be present along with P28 for effective plasmamembrane localization (Table 3).

Alterations to the C-terminus that result in truncation lead to NBTIbinding which is only slightly higher (e.g. T2 and T4) or equivalent tobackground (e.g. T8 Table 5) suggesting that important residues ormotifs for hENT1 localization to the plasma membrane lie within thisregion and supporting the conclusions derived from the confocal data(Table 5). The hydrophobicity of the FLFmotif appears to be importantsince non-polar to positive substitutions (mutants A–D) result in aconsiderable reduction in NBTI binding levels versus non-polar tonon-polar mutations (mutants G–I; Table 5). Substitution of thepositive arginine (R) with a negative glutamic acid (mutant E) led toapproximately half the level of control binding confirming theconclusions from the confocal data which suggested that the changein charge significantly affected localization to the plasma membrane(Table 5).

3.6. Functionality of hENT1 truncation and substitution mutants

Confocal microscopy and NBTI binding provide information on thelocation of GFP-hENT1 and the mutants. However neither assayaddresses the effect of these mutations on the functionality of proteinsat the plasma membrane. Therefore, to determine if mutants whichreach the plasma membrane, at least in part (Tables 2 and 4), wereindeed functionally equivalent to wildtype GFP-hENT1, we measureduptake of uridine, a well-established universal permeant, in Xenopuslaevis oocytes. Functionality of themutants is expressed as a percentageof the uptake seen by wildtype GFP-hENT1 (defined as 100%) and iscompared to uptake by the negative controls, pCDNA3.1+empty vectoror water injected oocytes, (which both showed similar results; definedas 0%).

Confocal and NBTI binding data suggest that the presence of theproline (in the PWN motif) ensures that at least half of the proteingets to the membrane. However, the transport data suggest that theseproteins, although correctly localized are functionally compromisedsince uptake is decreased (Table 3). This decrease is more than wouldbe anticipated on the basis of only half the protein reaching themembrane. The presence of the proline residue appears to contributeto the structural integrity of the protein, which enhances itsprocessing and targeting to the plasmamembrane while the presenceof the other two residues (WN) appear to provide more subtlecontributions to function.

Deletion mutants of the hENT1 C-terminus, which are partially orcompletely retained based on confocal and NBTI binding analyses,show reduced or non-existent uptake (e.g. T4 and T8<5%, Table 5).

Similar to our observations with confocal microscopy and NBTIbinding, we noted that transport activity of C-terminal substitutionmutants depends on the residue or region being substituted. Lysinesubstitutions, within the hydrophobic triplets (i.e. mutants A, D andL), show decreased transport activity, which correlates with reducedpresence at the plasma membrane. Of these lysine substitutionmutants, the most striking is mutant D (SKLFRAIV), which showsapproximately 41% of GFP-hENT1 at the plasma membrane based onconfocal microscopy and 48% based on NBTI binding, but virtually no

2333N.M.I. Nivillac et al. / Biochimica et Biophysica Acta 1788 (2009) 2326–2334

transport activity (Table 5). These data suggest that the firstphenylalanine in the FLF triplet is particularly important for function.This residue is conserved in many but certainly not all ENTs (Fig. 3B)and we noted that a conservative mutation of the phenylalanines toan alanine (mutants H and I; Table 5; which is present at the plasmamembrane in levels equivalent to wildtype hENT1) is less disruptiveto transport activity (i.e. ≤50%) suggesting that a non-polar residue(rather than specifically a phenylalanine) is important for function inthis region. Taken together with the confocal and binding data, ourfunctionality data suggest a role for charge in both function andlocalization.

Analysis of the contribution of the positively charged arginine inthe C-terminal region of hENT1 reveals that while a positive or non-polar amino acid (mutants K and J respectively) is sufficient to allowfor plasma membrane localization, introduction of these residuesresults in an overall reduction in transport activity (Table 5).However, conservation of the positive charge (mutant K; R H)retains transport activity which is closer to wildtype versus othermutations at this residue. Lastly, a positive to negative substitution inthis region (mutant E) shows a decreased transport activity (~45% ofwt) which correlates with a decreased presence at the plasmamembrane (50%).

4. Discussion

For effective nucleoside and nucleoside analog drug transport tooccur, hENT1 must be both localized and fully functional at theplasma membrane [11]. However, hENT1, like other membraneproteins, must first be folded correctly and be checked by ER qualitycontrol mechanisms so that it can be exported from the ER andcontinue to pass through the secretory pathway before reaching theplasma membrane [13,25]. Changes in the sequence of criticalregions of the protein that are responsible for any of these processescan cause disruptions in one or more of the processing stepsresulting in ER retention, misrouting or loss of function [37–39].Based on current models of hENT1 topology, it is likely that hENT1 isinserted into the ER membrane during translation in an orientationthat leaves the N-terminal PWN triplet in the membrane facing theER lumen and C-terminus extending into the ER lumen. Thus, whilemany other plasma membrane transporters possess targeting/trafficking motifs in the C-terminus, the predicted orientation ofhENT1 during processing would seem to preclude a role for the PWNtriplet or the C-terminus in an interaction with cytoplasmic proteinsinvolved in targeting and trafficking of the protein within the cell.Targeting and trafficking motifs thus have yet to be fully identified.Moreover, caution is required when interpreting data whichcorrelate localization patterns to specific mutations (motifs) sinceregions involved in the structural integrity of the protein, and thusfunction, must be retained for correct intracellular processing andtargeting to the plasma membrane. Function, therefore, relies oncorrect processingwhich is correlatedwith the structural integrity ofthe protein and different regions of a protein are likely to contributein varying ways (with the structural integrity of functionallyessential parts of the protein likely being particularly important incorrect processing).

Herewe identify two regions of hENT1 that are required for correctlocalization most likely due to their structural and functionalimportance. The PWN (or PYN) triplet is very highly conserved acrossENTs [10] and mutation of W29 has previously been shown to affectinhibitor sensitivity and nucleoside transport of hENT1 expressed inyeast [40]. Our findings also support a role of W29 in function, whichmay reflect the characteristic of tryptophan or tyrosine aromatic ringsto stack with purine and pyrimidine bases. Taken together these datasupport a central role of this motif in function but in addition, here wehighlight the essential role of P28 in maintaining the correct structureof this region of hENT1 for effective localization at the plasma

membrane in mammalian cells. Given the well-established role ofproline in maintaining protein structure [41], these findings demon-strate that structural integrity of hENT1 is essential in effectiveprocessing, localization and therefore function.

Since the C-terminus, like the PWN region, of hENT1 is ultimatelyextracellular, the retention seen with truncation of the C-terminus isunlikely to be due to disruption of targeting/trafficking motifs asdescribed for other transporters [i.e. 19,21]. Rather, our findings areconsistent with a role for this region in correct folding or structuralintegrity and are consistent with previous observations that demon-strate that alteration of a single residue within a critical motif orregion of a protein sequence can significantly alter protein folding[42]. The observation of partial retention of the T2 and T4 mutants isconsistent with the observation that some truncated proteins canescape the ER quality control and still localize to their intended site[43]. These data also suggest that key motifs possibly involved infolding (likely the FLF, hydrophobic triplet, motif) lie upstream of thelast 4 amino acids. Indeed, a previous study suggested that truncationsup to 4 amino acids had no effect on the location of hENT1 [35]suggesting that some truncated mutants can escape processingcheckpoints and find their way to the plasma membrane in certaincell types.

Similarly, we observed that disruption of charge (e.g. non-polar tolysine) in the hydrophobic triplets at the C-terminus leads to adisruption in the ability of hENT1 to localize to the plasma membranesupporting a potential role for the C-terminus in protein folding andpacking of the final transmembrane helices (while not specifically intargeting to the plasma membrane). It is possible that the phenyla-lanines in the FLF motif could act as docking sites for protein–proteininteractions as has been seen in the CFTR channel, the gonadotropin-releasing hormone receptor and the serotonin transporter [44–46].This is noteworthy since our findings show that the presence of asingle phenylalanine (plus two hydrophobic partners) is bothnecessary and sufficient for correct cell surface expression.

Disruption of the hydrophobicity in this region could then, in turn,disrupt the interaction of hENT1 with a major ER chaperone such asBiP, which aids in protein folding through interaction with hydro-phobic residues [13–15,47]. However, we cannot rule out thepossibility that the introduction of multiple lysines in the FLF regioncould cause retention due to the introduction of a di-lysine ERretention signal [48].

Taken together, our data suggest that, at the C-terminus, hENT1requires at least one phenylalanine and two hydrophobic residuesfollowed by a residue that can be positively charged or non-polar andfinally, a second hydrophobic triplet for effective plasma membranelocalization and transport activity. These data correlate with aproposed putative tertiary topology of the parasitic ENT1 homolog(LdNT2) which is based on a computational approach to definingstructure [33]. In this model both the PWN triplet and the C-terminus(the latter of which has been proposed to lie partially withintransmembrane domain 11) may contribute to the translocationpore. This model would therefore support the notion that these tworegions play a role in hENT1 function and may explain the reducedfunction seen with our plasma membrane localizing mutants (e.g.mutant D). Furthermore, if the PWN motif and C-terminal region doindeed line the translocation pore, they could provide a site forsubstrate recognition and/or binding.

Our findings suggest that localization combined with correctstructure dictate overall function of hENT1. Mutations in hENT1, thatpotentially affect structural integrity can also impact localization(presumably due to ER quality control mechanisms) and thusfunction. Thus factors involved in correct localization of hENT1 arecomplex and cannot simply be narrowed down to a single overlysimplified amino acid motif. This study provides the basis to furtheridentify factors such as interacting partners or proteinmotifs involvedin cellular processing, targeting or trafficking of hENT1.

2334 N.M.I. Nivillac et al. / Biochimica et Biophysica Acta 1788 (2009) 2326–2334

Acknowledgements

This work was supported by a Discovery Grant (RGPIN - 203397)from the Natural Sciences and Engineering Research Council ofCanada to IRC.

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