Cloning and functional characterization of a folate transporter from

the nematode, Caenorhabditis elegans

Krishnaswamy Balamurugan1, 2*, Balasubramaniem Ashokkumar1, 2*, Mustapha

Moussaif3, Ji Ying Sze3 and Hamid M. Said1, 2

1VA Medical Center, Long Beach, CA; 2Departments of Medicine (Nephrology) and

Physiology/Biophysics; 3Department of Anatomy and Neurobiology, School of Medicine, University of

California, Irvine, CA 92697

* Both authors contributed equally in this work.

Running title: Nematode folate uptake system

Address all correspondence to H. M. Said, VA Medical Center-151, Long Beach, CA 90822

Tel: (562) 826-5811; Fax: (562) 826-5675; Email: hmsaid@uci.edu

Page 1 of 51 Articles in PresS. Am J Physiol Cell Physiol (May 2, 2007). doi:10.1152/ajpcell.00516.2006

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

Two putative orthologs to the human reduced folate carrier (hRFC) folt-1 and folt-2 that

share a 40% and 31% identity, respectively, with the hRFC sequence have been identified in the

C. elegans genome. Functional characterization of the open reading frame of the putative folt-1

and folt-2 showed the folt-1 to be a specific folate transporter. Transport of folate by folt-1

expressed in a heterologous expression system showed an acidic pH-dependence, saturability

(apparent Km of 1.23 ± 0.18 µM), similar degree of inhibition by reduced and substituted folate

derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent Ki of 0.13 mM), and

inhibition by the anion transport inhibitors, e.g., 4,4'-diisothio-cyanatostilbene-2,2'-disulphonic

acid (DIDS). Knocking down (silencing) or knocking out the folt-1 gene led to a significant

inhibition in folate uptake by intact living C. elegans. We also cloned the 5’-regulatory region of

the folt-1 gene, and confirmed promoter activity of the construct in vivo in living C. elegans.

Using the transcriptional fusion construct (i.e., folt-1::GFP), the expression pattern of the folt-1

in different tissues of living animal was found to be highest in the pharynx and intestine.

Furthermore, the folt-1::GFP expression was developmentally and adaptively regulated in vivo.

These studies demonstrate for the first time the existence of a specialized folate uptake system in

C. elegans that has similar characteristics to that of the folate uptake process of the human

intestine. Thus, C. elegans provides a genetically tractable model that can be used for integrative

aspects of the folate uptake process in the context of the whole animal level.

Key words: C. elegans, integrative transport physiology, folate transport, transport regulation

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INTRODUCTION

Folate, a member of the B-class of water-soluble vitamins, is essential for normal cellular

functions and development. The vitamin acts as a coenzyme (one-carbon carrier) in a series of

metabolic reactions including the synthesis of precursors of DNA and RNA and the metabolism

of several amino acids including homocysteine (3, 36). Deficiency of this essential micronutrient

leads to a variety of abnormalities ranging from megaloblastic anemia to growth retardation and

neurological disorders. Humans and other mammals (as well as other multi-cellular eukaryotes)

are devoid of de novo biosynthesis of folate and therefore meet their folate requirement from

exogenous sources (5). The reduced folate carrier (RFC) is a major folate uptake system in

mammalian cells. The molecular identity of the RFC system has been delineated by cloning (21,

23, 40). The human RFC (hRFC) encodes a 591- amino acid plasma membrane protein that has a

predicted 12 transmembrane (TM) spanning domains, a long intracellular loop between trans-

membrane domains 6 and 7, and a short amino terminal (27 residues) and a long carboxy

terminal (138 residues) with both residing in the cytoplasm (9, 30). The 5’ flanking regulatory

regions of the RFC genes of human and a number of other mammalian species have been

characterized (19, 37). Other studies have shown that folate uptake in the mammalian cells to be

regulated by a variety of extracellular and intracellular factors (2, 16, 18, 25-27, 33). Much of

our recent knowledge on the physiology and biology of the folate uptake process was derived

from reductionist cell and molecular approaches. Much less is currently known about the

different parameters of the folate uptake process at the whole animal level. Such knowledge is

difficult to obtain in complicated organisms like mammals. The nematode Caenorhabditis

elegans has been used as an animal model in which to delineate molecular mechanisms of

complicated functions. (31-32). This animal model has a host of unique features that includes

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simple anatomy (it has a total of 959 highly differentiated cells), transparency, defined genomic

and tractable genetics, ease of maintenance and growth, a defined life cycle, and a short life-span

(2 to 3 weeks). In addition, many human physiological functions appear to have analogs in this

animal model, and many human genes have orthologs in the genome of this nematode, i.e.,

28.4% of the worm genome has one or more human orthologs and also 83% of worm proteins

have domains with significant similarity to human genes (24, 34). Furthermore, this animal

model allows great flexibility in manipulating physiological events and in performing certain

experiments that are otherwise difficult to perform at the whole animal level in vivo in more

complicated organisms (e.g., quantitative imaging and promoter analysis, can both be performed

at the whole animal level (31-32). Using this animal model, we have undertaken a series of

investigations into the integrative aspects of the folate uptake process. In this report, we present

our findings on the cloning of a C. elegans folate uptake system, the folt-1, the functional

characterization both in vitro and in vivo, and the effect of folt-1 on development and the

prevailing substrate level on different parameters of the folate uptake process at the level of

whole animal. Our results showed for the first time the existence of a specialized folate uptake

system (folt-1) in this species. This system appears to be similar in many ways to the folate

uptake process that operates in the human intestine, is being more active at acidic compared to

alkaline buffer pHs, having similar affinity to reduced and substituted folate derivatives, is

sensitive to the effects of the anti-inflammatory agent sulfasalazine, is inhibited by the anion

transport inhibitors DIDS and SITS, and has a similar apparent Km. In addition, the folt-1 system

was found to be expressed in different tissues of the nematode and appears to be

developmentally and adaptively regulated.

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MATERIALS AND METHODS

[3H]-Folic acid (specific activity, 26.2 Ci/mmol; radiochemical purity, 98.0%) was

obtained from Moravek Biochemicals (Brea, CA). Most of the fine quality analytical grade

chemicals, cell culture media and unlabeled compounds used in this investigation was obtained

from Sigma Chemical Co. (St. Louis, MO). Lipofectamine was purchased from Invitrogen

(Carlsbad, CA). The human retinal pigment epithelial (ARPE-19) cells were obtained from the

American Type Culture Collection, Inc. (Manassas, VA) and used in the studies to establish the

functionality of the cloned folts as done in similar previous investigations (6, 7, 41).

Nematode growth:

The wild-type nematode strain is Caenorhabditis elegans N2. For routine experiments,

the animals were maintained at 15-20°C on NGM agar plates, and Escherichia coli strain OP50

was used as the food source (4). Total RNA were prepared from worms by freezing the pelleted

worms in liquid nitrogen and grinding them in the presence of Trizol for total RNA preparation

as described by manufacturer’s protocol (Life Technologies, Rockville, MD).

Cloning of RFC-like transporters from C. elegans:

Three genes have been reported in the Worm genome (www.wormbase.org) as being

hRFC-like genes, C06H2.4, C50E3.11 and F37B4.7. These genes were named as folt-1, folt-2

and folt-3, respectively. Our own search of the C. elegans genome has confirmed that the C.

elegans genes 5L621 and 5D352 are indeed putative hRFC-like transporters sharing 40 and 31 %

identities with the hRFC but C50E3.11 did not show significant sequence homology with the

hRFC gene. Thus, we have renamed the putative folt-1 and folt-3 as folt-1 and folt-2, respectively

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to avoid confusion. To date no other gene with similarity to the hRFC gene was found. We also

searched for orthologs to the human folate receptor and the proton-coupled folate

transporter/heme carrier protein (PCFT/HCP1) in the worm genome but found none. We focused

our investigations on the putative folt-1 and folt-2. To clone these C. elegans RFC-like

transporters, we obtained an RT-PCR product using C. elegans poly (A)+ RNA and primers

designed on the basis of the predicted exonic sequences of these genes. Briefly, total RNA was

isolated from adult C. elegans using Trizol reagent. A pair of PCR primer specific for the

putative folt-1 and folt-2 genes was designed based on the sequences of the cosmids C06H2.4

and F37B4.7 (www.wormbase.org). Poly (A)+ RNA was used as template to perform the RT-

PCR employing the Superscript RT-PCR kit (Invitrogen, Carlsbad, CA) to synthesize first-strand

cDNA. To amplify the open reading frame (ORF) of the putative folt-1 and folt-2, we used the

following two gene-specific primers (for the folt-1, the forward primer was 5’-CCGCTC

GAGATGAGCTGGCGTACCAC-3’, and the reverse primer was 5’-CGGGATCCTCAATTTT

GGTCTAGAAAGACTG-3’; For the folt-2, the forward primer was 5’-ATGGAGCAATGGAA

AGTGATG-3’, and the reverse primer was 5’-TCAATTAGTACTCGTTTTGAAAAACCG-3’).

The PCR conditions used were: 95°C/10 min for 1 cycle; 95°C/30 sec., 54°C/1min., 72°C/3 min

for 40 cycles. A single PCR product was obtained for each ORF with an estimated size of ~1.2

and ~1.6 kb for the folt-1 and the folt-2 genes, respectively, as predicted by the distance between

these primers in each pair. The PCR products were gel-purified and sub cloned into pGEM-T

Easy Vector (Promega, Madison, WI). The molecular identity of the inserts was established by

sequencing (Laragen Inc, CA). The identified ORF were sub cloned into the mammalian

expression vector pLenti6/V5-Dest, again verified by sequencing, and then expressed in the

human retinal pigment epithelial cells (ARPE-19) to determine functionality.

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Functional expression of the cloned folt-1 and folt-2 in ARPE-19 cells using a lentiviral

expression system:

The ARPE-19 cells have been successfully used to functionally characterize cloned C.

elegans transporters (6, 7, 41). Using these cells, we expressed the putative folt-1 and the folt-2

using the lentivirus expression system as described previously (17, 22). Viral stocks were

prepared using folt-1 and folt-2-cDNA and the pLenti6/V5-Dest kit (Invitrogen Inc., Carlsbad,

CA) as per the manufacturer’s protocols. ARPE-19 cells (60-70% confluent) were transiently

transfected with 10 µl pLenti6/V5-DEST folt-1 or –2 cDNA virus/well of a 12-well plate in the

presence of polybrene (Fisher Scientific Inc., Tustin, CA), i.e., infected with a lentivirus at a

multiplicity of 5-10 plaque forming units/cell. The cells were incubated at 37°C for 72 h and

used for determination of transport activity. Cells transfected with vector alone without the

cDNA insert were used as the control to determine endogenous transport activity in these cells.

[3H]-Folate uptake was determined at 37°C in Krebs-Ringer buffer (in mM: 133 NaCl, 4.93 KCl,

1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 5.5; unless

otherwise stated). The [3H]-radioactivity taken up by the cells was determined by means of

scintillation counting. Protein content of cell digests was measured in parallel wells using a Bio-

Rad protein assay kit (Bio-Rad, Richmond, VA). Transport activity attributable to the expressed

folt was determined by subtracting folate uptake by vector transfected ARPE-19 cells from total

uptake by folt-1 and folt-2 expressing cells.

Folate uptake by the whole C. elegans:

The simplicity of the C. elegans body and its demonstrated ability to take up large and

small molecules from the exogenous sources (e.g., large dsRNAs; 8, 15) have led us to test the

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[3H]-folic acid uptake at the whole animal level. To obtain synchronized populations, we isolated

eggs from gravid adult animals that were treated with a hypochlorite/NaOH solution (14) to

isolate eggs. Eggs were incubated in M9 buffer and allowed to hatch overnight (approx. 12 h) at

room temperature. The resulting synchronized L1-stage worms were put on standard NGM

plates with feeding bacteria (~25oC) to develop to different stages. Worms at different

developmental stages were collected, washed with M9 buffer (containing 0.01% Triton X-100;

Sigma, St. Louis, MO), gently pelleted by centrifugation, and washed several times to remove

residual OP50 bacteria. They were then used in functional uptake assays at the whole animal

level, determining mRNA levels, and GFP expression pattern. To examine the effect of folt-1

gene specific RNA-interference (RNAi) on folate uptake we used the protocol described by

Timmons and Fire (35). The commercially available folt-1 gene specific RNAi feeder clone,

obtained from Open Biosystems (Huntsville, AL) was transformed into an E. coli strain, HT115

(DE3). The transformed bacteria culture was grown overnight and then induced with 1 mM

isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h, then seeded on NGM plates containing 1

mM of IPTG. To test the effect of folt-1RNAi, synchronized young adult C. elegans were

transferred to the plates, and the functional uptake assay was performed after 16 h of pre-

treatment. For the whole animal uptake studies, five age-synchronized individual young adult

nematodes were placed in a test-tube and were pre-incubated in Krebs-Ringer buffer for 20

minutes at room temperature. [3H]-Folic acid was then added and the reaction was terminated

after 5 min (initial rate; data not shown) by the addition of 1 ml of ice-cold Krebs-ringer buffer

followed by immediate placing of the animals on Millipore filters (0.22 micron) under negative

pressure followed by three times washing with ice-cold buffer. The filters with the animals on

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them (verified by microscope) were then transferred into vials containing scintillation fluid and

counted for radioactivity.

Semi-quantitative RT-PCR:

A RT-PCR assay (using folt-1-specific primers) was used to study the level of expression

of the endogenous folt-1 mRNA under different conditions [different developmental stages: early

larvae (L1-L3), young adult and adult; different level of exogenous folate]. Poly (A+) RNA was

used as a template to perform reverse transcription using an RT-PCR kit. The reverse

transcription was initiated with random oligos and carried out in a DNA thermal cycler (Light

Cycler PCR System, Bio-Rad Laboratories, Hercules, CA) as per the manufacturer’s procedures.

Reverse transcription was followed by real-time PCR in a single well format, in which the gene-

specific primers and the primers for the house keeping gene (β-actin) with their PCR mix (SYBR

green Kit, Qiagen, Valencia, CA) were combined separately at a predefined ratio. The PCR cycle

number was titrated according to the manufacturer’s protocol to ensure that the reaction was

within the linear range. The resultant PCR products were monitored during real-time and then

resolved in a 3.0% agarose gel for further confirmation. The steady state levels of folt-1 mRNA

were assessed from the relative cycle threshold (Ct) values during real-time PCR of the folt-1

specific RT-PCR product to the Ct values of the β-actin at each developmental stage/condition

and were calculated using a relative relationship method supplied by the iCycler manufacturer

(Bio-Rad).

Analyses of folt-1 knock out:

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To further our understanding of the role of folt-1 in folate uptake in C. elegans, we

obtained a deletion strain VC959 {Worm base deletion strain: VC959;tag-330(ok1460)V/nT1

[qIs51] (IV;V)) (C. elegans gene knockout consortium, Oklahoma Medical Research Foundation,

Oklahoma City, OK, USA)}, which has a deletion of ~1330 bp that covers part of the first exon

to the 3’-untranslated region of folt-1 (Fig. 7C) resulting in a complete removal of the coding

sequences which code for most of the transmembrane (TM) domains of folt-1. As per the KO

consortium report the homozygous ok1460 animals are sterile, but the basis of the sterility and at

what stage the sterility occurs is not clearly known. These animals do not produce offsprings, so

the ok1460 mutation has been balanced by nT1[qIs51]. The heterozygote strains can be

maintained and produce offsprings. The nT1[qIs51] animals are also not viable. So, the only

animals that were obtained on the plate were fertile heterozygotes and sterile ok1460

homozygotes. We furthered our studies with the sterile ok1460 homozygotes. We have selected

the homozygote worms on the basis of their phenotype (slow movement on the plate) and non-

GFP expression for uptake studies. The additional confirmations were performed by single worm

PCR (36) by using the following nested primers (Forwards 5’-TCTGCAACCGCAACGTATAA-

3’ and 5’-TTTCACCGGTCCATGAAAGT; Reverses 5’-TAACC-TTACTTCGACTT-3’ and 5’-

TCTT-GGCTCGGAGAA-3’) which produced a PCR product of ~2250 and 1040 bp for wild-

type and folt-1 KO animals, respectively.

Generation of transcriptional folt-1::GFP fusion construct and demonstration of its

promoter activity in vivo C. elegans:

To establish a genetically tractable model system in which to study the folate carrier, we

assessed the pattern of expression of the folt-1 in living nematodes. First, we cloned the 5’-

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regulatory region of the folt-1 gene using the sequence information deposited in Worm base

(accession number C06H2.4) for the folt-1 gene and flanking sequence and PCR. The 3’ end of

the adjacent up-stream gene was used as a 5’reference stop point. Two gene specific primers and

~50 ng of C. elegans genomic DNA were used to clone the entire (~1.4 kb) 5’-regulatory region

of the folt-1 gene (including its ATG). Then, we generated a transcriptional fusion construct that

contains 1.4 kb 5’-regulatory region of the folt-1 gene with the GFP reporter gene obtained from

the pPD 95.75 vector (gift from A. Fire, Carnegie Institution of Washington, Baltimore, USA) by

PCR (11). The fusion construct was verified by sequencing and was microinjected into the

syncytial gonad of adult wild-type C. elegans. The plasmid pRF4 containing the dominant

injection marker, Rol-6 (20) was coinjected as a transgenic marker. Transformants scored on the

basis of the roller phenotype behavior and GFP expression was observed under a fluorescence

microscope (Zeiss Axio plan II equipped with a fluorescence light source; Oberkochen,

Germany). Four independent lines carrying extra chromosomal arrays were obtained and all gave

similar patterns of GFP expression. The measurement of the GFP fluorescence was done at the

anterior, central, and posterior intestine of these transgenic lines. The transgenic animals were

recorded for their GFP fluorescence with fixed exposed time, and the intensities were measured

easily using Adobe Photoshop. The area of selection from each animal was identical for all of

our measurements.

Effect of exogenous folate level on expression of the folt-1 and on folate uptake by the whole

living C. elegans:

The effects of over-supplementation of the C. elegans with folate on the levels of

expression of the folt-1 and on the uptake of folate by the whole living C. elegans were

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examined. In these studies, wild-type and transgenic C. elegans expressing the transcriptional

construct folt-1::GFP were incubated for 24 h on nematode culture medium petridishes

supplemented with a larger dose of folate (1 mM) along with a E. coli OP50 bacteria lawn.

Findings with these animals were compared with findings in wild-type and transgenic nematodes

maintained in the absence of supplemented folate and fed bacteria that lacks the ability to

synthesize folate (Escherichia coli K12 MH828 and MH829 strains that are folA null mutants

(10); these strains were kindly provided by Dr. Muriel B. Herrington of Concordia University,

Montreal, Canada). The level of expression of the endogenous folt-1 mRNA, and in the level of

expression of the folt-1::GFP in transgenic C. elegans were then determined.

Statistical analysis:

All uptake studies were performed at least in triplicates on different occasions using

different batches of cells/nematodes and the data were expressed as mean ± SEM in mole/mg

protein/ unit time. Statistical analysis was performed using the Student’s t-test or one-way

Analysis of Variance followed by Tukey HSD test with statistical significance being set at 0.01

(p < 0.01). Kinetic parameters of the saturable folic acid uptake process were calculated using a

computerized model of the Michaelis-Menten equation as described previously by Wilkinson

(39). Uptake by the saturable process was determined by subtracting the diffusing component

(determined from the slope of the uptake line between a high pharmacological concentration of

folic acid of 1 mM and the point of origin) from the total uptake. Studies involving quantitative

PCR, analysis of promoter activity, and distribution of expression of folt-1 under different

conditions were performed on at least 20 to 30 different nematodes. For GFP analysis, 15-20

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age-synchronized nematodes were transferred to slides and were scored for fluorescence

intensity under identical times for comparison.

RESULTS

Cloning of the ORF of folt-1 and folt-2:

A search for genes in the C. elegans genome that have a reasonable degree of identity

with the hRFC resulted in the identification of two putative genes, folt-1 and folt-2. These genes

have 40% and 31 % identity with the hRFC, respectively. We cloned these two putative folate

transporters by RT-PCR using C. elegans poly (A)+ RNA and primers designed on the basis of

the predicted exon sequences of the individual gene. The size of the PCR products were ~1.23

and 1.6 kb for the folt-1 and folt-2, respectively, and their identities were confirmed by

sequencing. Of the two cloned sequences, only the folt-1 was found to have active folate

transporter activity (see below), and thus, we focused our characterization of this transporter. We

also did both 5’-RACE using Ambion’s FirstChoice RLM RACE Kit (Austin, TX) and 3’-RACE

using Invitrogen kit (Carlsbad, CA) to confirm the initiation and stop codons of folt-1. The

results confirmed the start and stop codons and showed lack of existence of any variance.

The deduced amino acid sequence of folt-1 in comparison with hRFC is given in Figure

1A. The cDNA of the folt-1 gene consisted of 1566 bp, of which 1233 bp represent the ORF.

This encodes for a protein of 410 amino acids with a predicted molecular mass of approximately

46.5 kDa. Hydropathy analysis (HMMTOP; www.enzim.hu/hmmtop1.1/server/hmmtop.cgi)

predicted the protein to have 10 TM domains with a long intracellular loop of 58 amino acids

between TM domains and 5 and 6 (Fig. 1B). This resembles the situation with the hRFC where a

large intracellular loop also exists. When the membrane topology was modeled, both the N- and

C-terminal ends were found to be directed toward intracellular side (Fig 1B). Direct studies

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however, are needed to establish the orientation of these terminals and the topology of the folt-1

transporter. The folt-1 polypeptide was predicted to carry two potential N- glycosylation sites

(Asn-36, Asn-260; NetNGlyc 1.0 server), five potential PKC phosphorylation sites (Ser-2, Ser-

186, Ser-192, Ser-212 and Ser-304) and two potential cAMP and cGMP-dependent protein

kinase phosphorylation sites (Lys-128 and Lys-226; NetPhos 2.0 server).

Functional characterization of the cDNAs of folt-1 and folt-2:

Functional identity of the cloned cDNAs of folt-1 and folt-2 was determined by

expressing them in a heterologous system of ARPE-19 cells followed by assaying for induction

in [3H]-folic acid uptake. Uptake of folic acid was investigated at 72 h following transfection and

data was compared to uptake by cells transfected with pLenti vector alone (control). First we

confirmed the expression of the folt-1 and folt-2 at the mRNA level in ARPE-19 cells (Fig. 2A).

Assay for [3H]-folic acid (16 nM) uptake then followed with results showing that the initial rate

of 3H-folic acid (16 nM) uptake was significantly (P < 0.01) (~6.5 folds) higher in cells

transfected with the full length folt-1 cDNA compared to control cells (8.96 ± 0.56 and 1.4 ±

0.15 fmol/mg protein/7 min for folt-1 transfected and control cells, respectively). Similar results

were obtained when a slightly shorter folt-1 protein (that lacks the last 53 AA that represents the

C-terminal tail and the last TM domain) was used, in that folic acid (16 nM) uptake was

significantly (P < 0.01) (8 folds) induced in cDNA transfected cells compared to control (Fig.

2B). The latter finding suggest that the C-terminal and last TM domain of folt-1 do not play a

role in the transport function of the carrier protein. Unless otherwise stated below, the functional

characterization studies were done using the slightly shorter form of folt-1.

Addition of unlabelled folic acid (50 µM) to the incubation medium led to a significant

(P < 0.01) inhibition in the initial rate of uptake of [3H]-folic acid (16 nM) by the cells

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transfected with shortened folt-1, while addition of the unrelated vitamins thiamin, biotin, and

ascorbic acid (all at 50 µM) failed to affect [3H]-folic acid uptake (Fig. 2B).

In contrast to folt-1, transfecting ARPE-19 cells with a cDNA for folt-2 failed to show

any increase in initial rate of folic acid (16 nM) uptake compared to controls, both at buffer pH

5.5 (2.19 ± 0.17 and 2.18 ± 0.05 fmol/mg protein/7 min, respectively) and buffer pH 7.4 (1.71 ±

0.07 and 1.69 ± 0.20 fmol/mg protein/7 min, respectively).

Effect of buffer pH and role of Na+ in folic acid uptake by the folt-1 system:

Effect of varying incubation buffer pH on the initial rate of folic acid (16 nM) uptake by

the induced carrier following transfection of ARPE-19 cells with cDNA of the shortened folt-1

was examined. The results showed an increase in folic acid uptake by the induced carrier as a

function of decreasing the incubation buffer pH; uptake was significantly (P < 0.01) higher at

buffer pH 5.5 compared to pH 7.4 (Fig. 3A). Similarly, uptake of folic acid (16 nM) by ARPE-19

cells transfected with full-length folt-1 was found to be significantly (P < 0.01) higher at pH 5.5

compared to pH 7.4 (8.96 ± 0.56 and 0.19 ± 0.03 fmol/mg protein/7 min at pH 5.5 and 7.4,

respectively). Incubation buffer pH 5.5 was used in all subsequent investigations.

The role of Na+ in folic acid uptake via the folt-1 system was examined by testing the

effect of isoosmotic replacement of Na+ with other monovalent cations (K+, Li+, Tris, choline

and NH4+) on the initial rate of folic acid (16 nM) uptake by the induced system in shortened

folt-1 expressing cells. The results showed the induced folic acid uptake to be similar in the

presence and absence of Na+ both at pH 5.5 (Fig. 3B) or at pH 7.4 (uptake of 1.04 ± 0.02, 1.07 ±

0.03, 1.11 ± 0.01, 1.08 ± 0.01, 1.18 ± 0.1, and 1.13 ± 0.2 fmol/mg protein/7 min, for the

incubation medium containing Na+, K+, Li+, Tris, choline and NH4+, respectively). We also

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examined the effect of pre-treating (for 30 min) the shortened folt-1 expressing ARPE-19 cells

with the Na+-K+-ATPase inhibitor, ouabain (0.5 mM) on the initial rate of folic acid (16 nM)

uptake. The results show the induced folic acid uptake to be similar in ouabain-pretreated and

control cells (10.86 ± 1.0 and 11.61 ± 0.562 fmol/mg protein/7 min, respectively).

Kinetic parameters of the induced folic acid uptake by folt-1 expressing ARPE-19 cells:

In this study, we examined the initial rate of folic acid uptake by the induced system in

shortened folt-1 expressing ARPE-19 cells as a function of increasing substrate concentration in

the incubation medium (0.01–10 µM). Uptake by the induced folic acid transport system

includes a saturable component (Fig. 4). The apparent Km and Vmax of the saturable uptake

component were then calculated as described in “Methods” and found to be 1.23 ± 0.18 µM and

7.28 ± 1.1 pmol/mg protein/7 min, respectively.

Effect of folate structural analogs, the anti-inflammatory drug sulfasalazine, and the anion

transport inhibitors DIDS and SITS on folic acid uptake by the induced system in folt-1

expressing cells:

The effect of the different concentrations of the reduced [(folinic acid or 5-

formyltetrahydro-folate (5-FTHF)] and substituted (methotrexate, MTX) folate structural analogs

on the initial rate of [3H]-folic acid (16 nM) uptake by the induced carrier in shortened folt-1

expressing ARPE-19 cells was examined at buffer pH 5.5. The results showed that both

structural analogues inhibit, in a concentration dependent manner, the uptake of [3H]-folic acid

by the induced system (Fig. 5). In both cases the inhibition was competitive in nature with

apparent inhibition constants (Ki) of 2.1 ± 0.3 µM and 1.6 ± 0.2 µM for 5-FTHF and MTX,

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respectively. Similarly, uptake of folic acid (16 nM) by ARPE-19 cells expressing the full-length

folt-1 was significantly (P < 0.01 for all) inhibited by 5 and 20 µM 5-FTHF (8.80 ± 0.60, 2.96 ±

0.40 and 2.40 ± 0.32 fmol/mg protein/7 min for control and in the presence of 5 and 20 µM 5-

FTHF, respectively) and by 5 and 20 µM MTX (8.80 ± 0.60, 2.48 ± 0.16 and 1.92 ± 0.16

fmol/mg protein/7 min for control and in the presence of 5 and 20 µM MTX, respectively).

We also examined the effect of different concentrations of the anti-inflammatory drug

sulfasalazine on the initial rate of folic acid uptake by the induced carrier in shortened folt-1

expressing ARPE-19 cells. Sulfasalazine is an anti-inflammatory drug that is widely used for the

treatment of inflammatory bowel disease (IBD) and is known to competitively inhibit the human

intestinal folate uptake process (42). The results showed sulfasalazine to cause a concentration-

dependent inhibition in the induced folic acid uptake process with the inhibition being

competitive in nature (apparent Ki of 0.13 ± 0.01 mM) (Fig. 6). Similarly, uptake of folic acid

(16 nM) by ARPE-19 cells expressing the full-length folt-1 was significantly (P < 0.01)

inhibited by 0.25 and 0.5 mM sulfasalazine (8.80 ± 0.60, 3.44 ± 0.90 and 2.88 ± 0.24 fmol/mg

protein/7 min for control and in the presence of 0.25 and 0.5 mM sulfasalazine, respectively).

We have tested the effect of the anion transport inhibitors DIDS and SITS (inhibitors of

the human intestinal folate uptake process; 28-29) on the initial rate of folic acid uptake by

induced carrier in shortened folt-1 expressing ARPE-19 cells. The results showed both

compounds to cause a significant (P < 0.01) inhibition in folic acid (16 nM) uptake (8.01 ± 0.8,

0.98 ± 0.01 and 0.89 ± 0.05 fmol/mg protein/7 min, for control, and in the presence of DIDS and

SITS, respectively).

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Confirmation of functionality of the folt-1 as a folate transporter in vivo: Effect of folt-1

knock down (silencing) and knock out:

To establish the functionality of the folt-1 as a folate transporter in vivo in C. elegans, we

used two approaches. In the first, we examined the effect of specific folt-1 gene silencing on

whole animal folate uptake, with the use of a gene specific RNAi feeder clone. In these studies,

wild-type animals were fed with E. coli expressing the folt-1RNAi plasmids for 16 h; control C.

elegans were fed bacteria without the RNAi. Real-time PCR analysis was then performed on

samples from these animals with the results showing a significant (P < 0.01) reduction in mRNA

levels of folt-1 in the RNAi fed animals compared to controls (Fig. 7A). This reduction appears

to be specific for folt-1 as no change in the level of expression of the house keeping gene β-actin

was observed. Next, we examined the functional consequences of the folt-1 silencing on the

whole animal folate uptake [5 min; uptake by the whole animal was linear for up to 20 min (data

not shown)]. The results showed a significant (P < 0.01) reduction in folate (16 nM) uptake in

RNAi fed compared to control animals (Fig. 7B). Uptake of the unrelated ascorbic acid, on the

other hand, was similar in the two animal groups (Fig. 7B).

In the second approach, we used a folt-1 deletion strain (Fig. 7C) of the C. elegans and

performed similar functional folate uptake studies. Consistent with the KO consortium report, we

found the homozygotes of this mutant to be defective in reproduction (sterile) and displayed very

slow (sluggish) movement. The identity of the strain was confirmed by PCR (38) and by the

phenotypic characteristics. Wild-type animals showed a PCR product of ~2250 bp, whereas the

folt-1-/- KO animals showed a deletion product of ~1040 bp (Fig. 7D).

Using the homozygote folt-1-/- deletion strains, we examined folate uptake by these

animals and compared the findings to that in wild-type animals of identical stages

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(synchronized). The results showed a significantly (P < 0.01) lower folic acid (16 nM) uptake in

the homozygote folt-1-/- KO animals compared to wild-type (Fig. 7E). Uptake of the unrelated

biotin, on the other hand, by the homozygous folt-1-/- KO animals was similar to that of wild-

type animals (Fig. 7E).

Analyses the expression pattern of folt-1:

To study the pattern of expression of the folt-1 gene in the whole living nematode, a

transcriptional folt-1::GFP fusion was constructed (see “Method”), then used to generate

transgenic nematodes expressing this transgenic construct. In this transcriptional construct, the

expression of the GFP would be indicative of expression pattern of the folt-1 gene. The results

showed expression of the GFP in different C. elegans tissues thus establishing promoter activity

of the cloned genomic fragment. Expression was consistently higher in the pharynx and the

posterior part of intestine; it was also observed in the body wall muscles, head muscles and vulva

muscles of these transgenic animals (Fig. 8).

Developmental regulation of the folt-1 expression:

To study the effect of C. elegans development on the level of expression of the folt-1

gene, total RNA was isolated from whole synchronized C. elegans at different developmental

stages (larva 1, larva 2, young adult, and adult). Message level of the folt-1 was then determined

by means of quantitative real-time PCR (Fig. 9A) using poly (A+) RNA samples. Real-time PCR

analysis of β-actin transcripts from the same samples served as an internal control in these

experiments. There was a gradual decrease in the expression levels of the folt-1 mRNA with

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development with the decrease reaching a significant (P < 0.01) level when the animal reached

to the adult stage.

The effect of development on the level of expression of the folt-1 gene was also

determined in vivo using transgenic nematodes expressing the transcriptional construct folt-

1::GFP. In these studies we focused on studying the effect of development on level of

expression of folt-1 (i.e., on fluorescence intensity of the GFP) in the intestine due to our long-

standing interest in intestinal physiology. The results showed the level of GFP expression

(fluorescence intensity) in the intestine to be significantly (P < 0.01) higher at the early larva

stage compared to latter stages (Fig. 9B).

Effect of exogenous folate level on parameters of folate uptake and on the level of folt-1

expression:

The effect of maintaining (for 72 h) C. elegans in a culture medium containing high

concentrations of folic acid (1 mM) on the level of expression of the folt-1 mRNA and other

parameters of folate uptake was examined. Comparison was made to data from C. elegans

maintained in regular culture medium (no folate supplementation) and fed E. coli that lacks the

ability to synthesize folate (we used this type of E. coli in order to further minimize the level of

exogenous folate that is available to the animals so that a clearer comparison can be made). The

latter animals were considered as controls in this study. Results of the quantitative PCR assay

showed the level of the folt-1 mRNA to be significantly (P < 0.01) lower in the C. elegans

maintained in folate over-supplemented culture medium compared to the controls (Fig. 10A).

In a related study, we examined and compared the effect of maintaining C. elegans in

folate over-supplemented medium on short-term [3H]-folic acid (16 nM) uptake by the whole C.

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elegans. The results showed the uptake to be significantly (P < 0.01) lower in nematodes

maintained in folate over-supplemented medium compared to those of control (Fig. 10B).

However, the uptake of the unrelated vitamin, biotin (9.6 nM) by the whole C. elegans, on the

other hand, was similar in the two animal groups (0.25 ± 0.01 and 0.23 ± 0.05 fmol/5 animals/5

min, respectively). In another study we examined the effect of maintaining the folt-1 KO animals

on [3H]-folic acid (16 nM) uptake by the whole C. elegans. Results were compared to folic acid

uptake by KO animals maintained in control medium. The results showed similar uptake by the

folt-1 KO C. elegans under two folate conditions (0.02 ± 0.001 and 0.019 ± 0.004 fmol/5

animals/5 min, in folt-1 KO animals maintained in folate over supplemented and control

medium, respectively).

To link the above observed changes in the level of expression of the folt-1 mRNA and in

folic acid uptake upon folate over-supplementation with possible transcriptional regulatory

events, transgenic nematodes carrying the folt-1::GFP transcriptional construct were maintained

under folate over-supplemented and control conditions followed by determination of GFP

fluorescence intensity in the intestine. There was a significantly (P < 0. 01) lower level of GFP

expression in the intestine of the folate over-supplemented transgenic animals compared to

control (Figs. 11A, B and C).

DISCUSSION

Our aims in these investigations were to establish the nematode C. elegans as an in vivo

model system with which to study integrative aspects of folate uptake at the whole animal level

in vivo and to characterize the folate uptake system expressed in this animal species and study its

regulation. The suitability of C. elegans as a model system in such investigations has been well

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established with important findings being made which are of relevance to those found in higher

mammals like humans (31-32). In these investigations we used the information available in the

Worm database and our own search to identify two putative hRFC-like genes that were named as

folt-1 and folt-2. The products of these genes shared 40% and 31% identity with the hRFC,

respectively. To examine the possible role of these genes in folate uptake, we cloned the cDNAs

of both putative folate like transporters and tested their functionality as folate transporters in a

heterologous cellular system. Our results indicated that only the folt-1 to be the active folate

transporter. Comparing the protein sequence of the folt-1 with that of hRFC showed similarities

between the two sequences to be mainly localized to regions in TM domains 2, 3, 4, 5, 6 and 11

of hRFC. Interestingly, some of these hRFC domains or residues are involved in substrate

binding and functionality (12, 13). Further functional characterization of folt-1 showed that while

unlabelled folic acid inhibited [3H]-folic acid uptake by the induced system, neither the

structurally unrelated biotin nor ascorbic acid was found to affect the folate uptake. The later

findings indicate the specificity of the folt-1 as a folate uptake system. The induced folate uptake

in folt-1 expressing ARPE-19 cells was found to be saturable with an apparent Km of 1.23 ± 0.18

µM, pH (but not Na+)-dependent with a markedly higher uptake at acidic compared to neutral

and alkaline buffer pHs. Uptake by the induced carrier was also sensitive to the inhibitory effect

of the reduced folate structural analog 5-FTHF and the substituted analog MTX. Interestingly

the inhibition of [3H]-folic acid uptake by 5-FTHF and MTX was competitive in nature with an

apparent Ki of 2.1 and 1.6 µM, respectively. The similar apparent Km for folic acid uptake and

the inhibition constants of 5-FTHF and MTX suggest that these substrates have similar affinities

for the folt-1. The acidic pH dependence of the folate uptake process by folt-1 and its similar

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affinities to oxidized, reduced and substituted folate derivatives are similar to the characteristics

of the folate uptake process of the human intestine (28).

An interesting observation was the ability of the anti-inflammatory agent sulfasalazine to

competitively inhibit the induced folic acid uptake in the folt-1 expressing ARPE-19 cells.

Sulfasalazine is an anti-inflammatory agent that is widely used in the treatment of IBD and has

been shown to competitively inhibit folate uptake in the human intestine (42). In addition and as

seen with the human intestinal folate uptake process (28-29), the anion transport inhibitors DIDS

and SITS were both found to be strong inhibitors of folic acid uptake by the induced system in

the folt-1 expressing ARPE-19 cells. The latter two findings further indicate the similarity

between the functionality of the folate uptake process mediated by folt-1 in C. elegans and that

of the human intestinal folate uptake process.

To confirm the functionality of the folt-1 system in vivo, we utilized two different

approaches. In the first approach, we examined the effect of knocking down (silencing) the folt-1

gene with the use of gene-specific RNAi on folate uptake. In C. elegans, the RNAi -mediated

gene silencing process is so robust that exposure of the animals to RNAi in their environment is

sufficient to induce genetic interference (35). The results showed that silencing the folt-1 gene

leads to a substantial reduction in folate uptake compared to the control (Fig. 7B). In the second

approach, we used folt-1 knock out C. elegans and examined folate uptake; results were

compared to that of identical stage wild-type animals. The folt-1 knock out worms was obtained

from the deletion strain VC959. First, we selected the homozygote animals based on phenotype

and PCR data (genotyping). We then used these animals in folate uptake studies and compared

the findings to those of identical aged wild-type worms. The results showed folate uptake to be

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severely inhibited in the KO worms compared to controls. These findings collectively suggest

the critical role played by folt-1 in folate uptake in C. elegans in vivo.

To gain insight into the transcriptional regulation of the folt-1 gene, we cloned the 5’-

regulatory region of the gene, and fused the cloned genomic fragment to the GFP reporter gene.

Promoter activity of the cloned folt-1 genomic fragment was demonstrated in vivo by generating

transgenic worms expressing the folt-1::GFP which showed expression of the GFP in the living

animals. This study, in addition to demonstrating promoter activity of our cloned genomic

fragment also provided important information on the pattern of expression of the folt-1 in

different tissues of the intact whole C. elegans in vivo since expression of the GFP was driven by

the folt-1 promoter. The results showed the highest level of expression to be in two organs of the

digestive system, namely the pharynx and the (posterior portion of) intestine of the transgenic

animals. While the intestinal area is composed of highly differentiated epithelial cells, the

pharynx area contains different cell-types including epithelial cells, muscle cells, and secretory

glands (1). The latter cell-type is believed to be involved in the secretion of digestive enzymes

(1). The high level of expression of the folt-1 in the cells of digestive system raises the possibility

of its involvement in micro-nutrient absorption in this organism. Further studies are, however,

needed to confirm this suggestion.

We also investigated possible developmental regulation of folt-1 mRNA expression in

wild- type and in transgenic C. elegans expressing the transcriptional folt-1::GFP construct. The

results showed the highest level of expression of the folt-1 mRNA to be in the larva 1 stage but

the expression declined with maturation. This pattern of decline was also observed in the

intestine of transgenic animals expressing the transcriptional construct folt-1::GFP, thus,

confirming the in vitro observations. The observation that the folt-1 is developmentally regulated

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is similar to the observations reported for mammalian RFC, whose expression in the gut was

shown to be developmentally regulated and in a similar manner (2, 26).

Possible adaptive regulation of the folt-1 expression and function was investigated using

both wild-type and transgenic animals carrying the folt-1::GFP construct. Maintaining C. elegans

in culture medium over-supplemented with high pharmacological doses of folic acid was found

to lead to significant decrease in the level of mRNA expression of the folt-1. This decrease was

associated with a specific decrease in the level of folic acid uptake by wild-type C. elegans.

However, such a regulation by external folate level was not observed with KO worms, further

supporting the present data on the important role played by the folt-1 gene in folate uptake

process in C. elegans. These changes indicate that uptake of folate by the folt-1 is adaptively

regulated by exogenous substrate level. The observation of a decreased expression of folt-1::GFP

in the intestine of the C. elegans maintained in folate over-supplemented medium compared to

control suggest that transcriptional regulatory mechanism(s) may (at least in part) be involved in

mediating the observed adaptive response. Again, these observations are similar to those reported

with mammalian RFC in the intestine upon changing extra-cellular folate levels (27, 33, and

unpublished observations).

In summary, results of these investigations have identified for the first time the existence

of a functional and specialized folate uptake system in the nematode C. elegans and showed the

system to be similar to that of the human intestinal folate uptake process in being acidic pH-

dependent, having similar affinity to oxidized, reduced and substituted folate derivatives, is

sensitive to the inhibitory effects of the anti-inflammatory agent sulfasalazine, and is inhibited by

the anion transport inhibitors DIDS and SITS. In addition, functionality of this system was

confirmed in vivo by gene knock down and knock out approaches. Furthermore, the folt-1

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appears to be expressed in different tissues of the C. elegans (including the intestine) and that its

expression is regulated during development and by substrate level in the culture medium. These

studies establish the suitability of the C. elegans as a model for detailed investigations into

integrative aspects of the folate uptake process at the whole animal level.

ACKNOWLEDGEMENTS

We would like to express our sincere thanks to Dr. Kevin Strange (Vanderbilt University,

Tennessee) and Dr. Keith Nehrke (University of Rochester, New York) for their valuable

discussions and advices. We would also like to thank Dr. Bin Liang for his technical assistance

with microinjection and interesting discussions.

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FOOTNOTES

• This study was supported by grants from the Department of Veterans Affairs and the

National Institutes of Health (DK58057 and DK075348 to H.M.S) and (MH64747 to J.S).

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FIGURE LEGENDS

Figure 1. (A) Comparison of amino acid sequences of C. elegans folt-1 and human RFC.

Alignment (www-archbac.u-psud.fr/genomics/multalin.html) of the amino acid

sequences of folt-1 (upper panel) with hRFC (lower panel). The symbols ($, % and #) denote

anyone of these residues (LM, FY and NDQEBZ, respectively). The symbol (∗) denotes the

length of the shortened folt-1 protein (357 AA). Transmembrane domains are in shaded boxes.

(B) Schematic representation of the predicted secondary structure of the folt-1 protein

determined by HMMTOP (www.enzim.hu/hmmtop1.1/server/hmmtop.cgi). The protein is

predicted to have ten transmembrane domains. The symbol (∗) denotes the length of the

shortened folt-1 protein (357 AA).

Figure 2. (A) Expression of the folt-1 and 2 at the mRNA level following transfection into

ARPE-19 cells.

Cells were transfected with folt-1 and 2 as described in “Methods”. Total RNA isolated

from these cells was analyzed by quantitative real-time PCR for the presence of folt-1 and 2

transcripts. Data were normalized relative to the house-keeping gene, β-actin.

(B) Functional identification of the cloned shorter form of folt-1 as a specific folate

transporter in ARPE-19 cells.

Confluent monolayers of ARPE-19 cells expressing the folt-1 were incubated with [3H]-

folic acid (16 nM) for 7 min (initial rate) at 37°C in Krebs-Ringer buffer pH 5.5 in the absence

(control) and presence of 50 µM of unlabeled folic acid, thiamin, biotin or ascorbic acid. Data

are mean ± SEM of at least three separate uptake determinations.

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Figure 3. Effect of incubation buffer pH (A) and Na+(B) on folate uptake by ARPE-19 cells

expressing the shortened folt-1.

Confluent monolayers of ARPE-19 cells expressing the shortened folt-1 were incubated

for 7 min (initial rate) at 37°C in Krebs-Ringer buffer. [3H]-folic acid (16 nM) was added to the

incubation medium at the onset of incubation. Data are mean ± SEM of at least 3 separate

uptake determinations.

Figure 4. Initial rate of folic acid uptake as a function of concentration by ARPE-19 cells

expressing the shortened folt-1.

Confluent monolayers of the ARPE-19 cells expressing the shortened folt-1 were

incubated in the presence of different concentrations of [3H]-folic acid. Uptake was determined

following 7 min of incubation in Krebs-Ringer buffer pH 5.5 at 37°C. Uptake by the saturable

component was calculated as described in “Methods”. Data are means ± SE of at least 3 separate

uptake determinations. When not shown, SE bars are within the symbol size.

Figure 5. Dixon plot for the effect of the folate structural analogues folinic acid (A) and

methotrexate (MTX) (B) on uptake of [3H]-folic acid by ARPE-19 cells expressing the

shortened folt-1.

Confluent monolayers of ARPE-19 cells expressing the shortened folt-1 were incubated

for 7 min (initial rate) at 37°C in Krebs-Ringer buffer pH 5.5. [3H]-Folic acid [0.1 (o) and 1 (●)

µM) and different concentrations of 5-formyltetrahydrofolic acid (folinic acid) and methotrexate

(MTX) were added at the onset of incubation. Data are mean ± SE of at least 3 separate uptake

determinations.

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Figure 6. Dixon plot for the effect of sulfasalazine on folic acid uptake by ARPE-19 cells

expressing the shortened folt-1.

Confluent monolayers of ARPE-19 cells expressing the shortened folt-1 were incubated

for 7 min (initial rate) at 37°C in Krebs-Ringer buffer pH 5.5. [3H]-Folic acid [0.1 (o) and 1 (●)

µM] and different concentrations of sulfasalazine were added at the onset of incubation. Data

are mean ± SE of at least 3 separate uptake determinations.

Figure 7. (A) Effect of silencing the folt-1 gene with RNAi on mRNA levels of the

endogenous folt-1.

Total RNA was isolated from the control and folt-1 gene specific RNAi fed animals and

was analyzed by quantitative real-time PCR for the level of folt-1 transcripts as described in

“Methods”. Data were normalized relative to the house-keeping gene, β-actin.

(B) Effect of silencing the folt-1 gene with RNAi on folate uptake by C. elegans.

Age synchronized young adult wild-type C. elegans fed with E. coli expressing folt-1

RNAi plasmids or with normal bacteria (control) for 16 h as described in “Methods”. Uptake by

the whole animal (at 21°C) was examined by incubating (5 min) in Krebs-Ringer buffer pH 5.5

in the presence of [3H]-folic acid (16 nM) or [14C]-Ascorbic acid (30 µM). Each data point

represents the mean ± SE of 3 to 6 separate uptake determinations from different batches of

animals. Five nematodes were added to each set of experiments. Notice the significant (p <

0.01) inhibition in carrier-mediated folate uptake but not in the uptake of the unrelated ascorbic

acid in the folt-1 RNAi fed C. elegans compared to controls.

Page 35 of 51

36

(C) Intron-exon organization of the folt-1 showing the deletion region in the KO

animals.

Exons are indicated in boxes. The KO consortium produced folt-1 gene knock-out allele,

is shown at the bottom (ok1460; dark box). The deletion covers most of coding regions and

functional sites including the predicted TM domains (2-10) of the folt-1. Figure adopted from

http://worm.imbb.forth.gr/db/gene/gene?name=WBGene00007388;class=Gene.

(D) Confirmation of folt-1 homozygote knock-outs from the deletion strain VC959 C.

elegans.

Single worm genomic DNA was analyzed by PCR for the presence of the full-length

wild-type folt-1 (~2250 bp) and the knock out folt-1 (~1040 bp) fragments. Lane 1 indicates the

DNA molecular weight markers. Lanes 2-5 include PCR products that indicate homozygote KO

folt-1, and lanes 6-9 include the PCR product indicate the wild-type folt-1.

(E) Effect of knocking out the folt-1 on folate uptake by the C. elegans.

Wild- type and folt-1 knockout C. elegans were incubated (at 21oC) for 5 min in Krebs-

Ringer buffer pH 5.5 in the presence of [3H]-folic acid (16 nM) or [3H]-biotin (9.6 nM). Each

data point represents the mean ± SE of 3 to 6 separate uptake determinations from different

batches of animals. Five nematodes were added to each incubation reaction. Notice the

significant (p < 0.01) inhibition in folate uptake, but not in the uptake of the unrelated biotin, in

the knockout C. elegans compared to wild-type.

Page 36 of 51

37

Figure 8. Activity of the folt-1 promoter in vivo.

A representative transgenic C. elegans showing the expression of full length promoter

fusion construct, folt-1::GFP. The construct was generated by fusing the folt-1 promoter with

pPD95.75 vector (see “Methods”). Transgenic animals carrying this transcriptional fusion

construct were maintained until F4 generation and scored for the level of GFP expression for the

next few generations. Bottom panel represents the bright field image of the same worm.

Figure 9. Effect of development on the level of folt-1 expression.

(A) Effect of development on the level of expression of the endogenous folt-1 mRNA.

PolyA+ RNA samples were isolated from synchronized C. elegans at different

developmental stages and were used for the real-time PCR analysis as described in “Methods”.

Data were normalized relative to a house-keeping gene, β-actin, and calculated using a relative

relationship method supplied by the manufacturer (Bio-Rad).

(B) Effect of development on the levels of expression folt-1::GFP in the intestine of

living transgenic C. elegans.

Synchronized C. elegans at different developmental stages were monitored for their GFP

fluorescence intensities in the intestinal area. The pixel values were recorded and compared

between each groups. Data are from at least 20 animals from each stage and were done at three

different occasions.

Figure 10. Effect of folate levels in culture media on endogenous folt-1 mRNA level (A) and

on [3H]-folic acid uptake (B) by whole living young adult C. elegans.

Page 37 of 51

38

Young adult wild-type C. elegans were maintained for 24 h in a culture medium

containing high (over-supplemented) and low (control) concentrations of folate as described in

“Methods”. (A) Poly A+ RNA isolated from these young adult nematodes under the above

mentioned conditions, were used for the real-time PCR analysis. Data were normalized relative

to a house keeping gene, β-actin. (B) [3H]-Folic acid (16 nM) uptake was examined following 5

min incubation at 37oC in Krebs-Ringer Buffer pH 5.5 as described in “Methods”. Each data

point represents the mean ± SE of 3 to 6 separate uptake determinations from different batches of

animals. Five young adult nematodes were used in each experiment.

Figure 11. Effect of maintaining C. elegans in growth media over-supplemented with folate

on the activity of the folt-1 promoter in living animals.

Transgenic C. elegans expressing the folt-1::GFP were maintained in control (A) and

folate over-supplemented (B) conditions. Intensity of the GFP fluorescence in the intestine of

transgenic animals was determined as described in “Methods”. (C) Represents comparison of the

level of folt-1::GFP expression in animals maintained under two different folate conditions.

Page 38 of 51

39

Folt-1 1 MSWRTTTAILCLYGAVKEFRPATPFLTPFLASPEKNIThRFC MVPSSPAVEKQVPVEPGPDPELRSWRHLVCYLCFYGFMAQIRPGESFITPYLLGPDKNFTConsensus ......................mSWRhltaiLCfYGama#fRPaepFiTP%LagP#KNfT

61Folt-1 LDELYSQVYPYWTYSYMLALIPMFILTDILRYKPIVMIEAIGLVATWALLVFGKGVWQMQhRFC REQVTNEITPVLSYSYLAVLVPVFLLTDYLRYTPVLLLQGLSFVSVWLLLLLGHSVAHMQConsensus l##ltn#itPvlsYSY$aaLiPmFiLTDiLRYkPil$i#aigfVatWaLLlfGhgVahMQ

121Folt-1 IMQVSFGVASAAEIAYYSYIYSIVDRKHYKRATSYIRAAALMGKLVAFGLGQTLISTHTShRFC LMELFYSVTMAARIAYSSYIFSLVRPARYQRVAGYSRAAVLLGVFTSSVLGQLLVTVGRVConsensus iM#lf%gVamAAeIAYsSYI%SiVdpahYkRaagYiRAAaL$GkftafgLGQlListgrs

181Folt-1 DYLVLNQISLGAVCLVTIIAIFLPRVKS EKAKVSMRAHEIVEQQTVESVESVQTPKhRFC SFSTLNYISLAFLTFSVVLALFLKRPKRSLFFNRDDRGRCETSASELERMNPGPGGKLGHConsensus d%ltLNqISLaalcfstiiAiFLkRpKr....#kadrgmcaheasEl#rm#pgeggklgh

241Folt-1 AVKMSYTREYFKKISEELQICSKNQELLKWSLWWALASCGVYQVQNYTQSLWKELQ NNhRFC ALRVACGDSVLARMLRELGDSLRRPQLRLWSLWWVFNSAGYYLVVYYVHILWNEVDPTTNConsensus AlkmacgdevfakileELgdclknp#LlkWSLWWafaSaGvYlVqnYthiLWkEl#..nN

301Folt-1 PDDVANGVVEFVNTALGAFLSLFIHHLSIDWT LRYVLRAhRFC SARVYNGAADAASTLLGAITSFAAGFVKIRWARWSKLLIAGVTATQAGLVFLLAHTRHPSConsensus padVaNGaa#aanTaLGAflSfaagflkIdWa................Lr%lLaa.....

361 Folt-1 NVAKELSSNNHGLIFGCNTFVAVCLQSLLTLVVVDhRFC SIWLCYAAFVLFRGSYQFLVPIATFQIASSLSKELCALVFGVNTFFATIVKTIITFIVSDConsensus .........................#iAkeLSk#lcaLiFGcNTFfAtclksiiTfiVsD

421 ∗Folt-1 SRFLHLDIRTQFVIYSGYFALVASIFAFFFMISLFSKSSNAH TAQTTYEATNEIQhRFC VRGLGLPVRKQFQLYSVYFLILSIIYFLGAMLDGLRHCQRGHHPRQPPAQGLRSAAEEKAConsensus sRfLgLdiRkQFqiYSgYFailaiI%affaMidgfrhcqnaH.....pAQglreAa#Eia

481Folt-1 EETVFLDQNhRFC AQALSVQDKGLGGLQPAQSPPLSPEDSLGAVGPASLEQRQSDPYLAQAPAPQAAEFLSPVConsensus a#alfl##k...................................................

Folt-1 541hRFC TTPSPCTLCSAQASGPEAADETCPQLAVHPPGVSKLGLQCLPSDGVQNVNQConsensus ...................................................

Figure 1A

Page 39 of 51

40

Predicted secondary structure

Figure 1B

1 2 3 4 5 6 7 8 9TMD

NH+

COO-

Asn 36Asn 260

10

Membrane

OUT

IN

*

1 2 3 4 5 6 7 8 9TMD

NH+

COO-

Asn 36Asn 260

10

Membrane

OUT

IN

*

Page 40 of 51

41

A

B

Figure 2

0

5

10

Contro

lfo

lt-1

mR

NA

exp

ress

ion

over

β-a

ctin

(in

fold

s)

folt-

2

0

5

10

Contro

lfo

lt-1

mR

NA

exp

ress

ion

over

β-a

ctin

(in

fold

s)

folt-

2

Cells expressing shortened folt-1

Contro

l Vec

tor

Folic

acid

Thiam

ine

Biotin

Ascor

bic a

cid

3 [H

] Fo

lic A

cid

upta

ke(f

mol

/mg

prot

ein/

7 m

in)

0

12

No ad

ditio

n

Cells expressing shortened folt-1

Contro

l Vec

tor

Folic

acid

Thiam

ine

Biotin

Ascor

bic a

cid

3 [H

] Fo

lic A

cid

upta

ke(f

mol

/mg

prot

ein/

7 m

in)

0

12

No ad

ditio

n

Page 41 of 51

42

A

B

Figure 3

0

12

5.0

5.5

6.0

6.5

7.0

7.4

8.0

3 [H

]-Fo

lic

acid

upt

ake

(fm

ol/m

g pr

otei

n/7

min

)

0

12

5.0

5.5

6.0

6.5

7.0

7.4

8.0

3 [H

]-Fo

lic

acid

upt

ake

(fm

ol/m

g pr

otei

n/7

min

)

0

12

Contro

l (Na+

)

K+

Li+

TrisCho

line

NH 4+

3 [H

]-Fo

lic

acid

upt

ake

(fm

ol/m

g pr

otei

n/7

min

)

0

12

Contro

l (Na+

)

K+

Li+

TrisCho

line

NH 4+

3 [H

]-Fo

lic

acid

upt

ake

(fm

ol/m

g pr

otei

n/7

min

)

Page 42 of 51

43

Figure 4

0

5

10

3 [H

]-Fo

lic

acid

upt

ake

(pm

ol/m

g pr

otei

n/7

min

)

5 10

Folic acid [µM]

Km= 1.23 ± 0.18 µMVmax= 7.28 ±1.1 pmol/mg protein/7 min

0

5

10

3 [H

]-Fo

lic

acid

upt

ake

(pm

ol/m

g pr

otei

n/7

min

)

5 10

Folic acid [µM]

0

5

10

3 [H

]-Fo

lic

acid

upt

ake

(pm

ol/m

g pr

otei

n/7

min

)

5 10

Folic acid [µM]

Km= 1.23 ± 0.18 µMVmax= 7.28 ±1.1 pmol/mg protein/7 min

Page 43 of 51

44

A

B

Figure 5

1.5

-20 -10

Methotrexate [µM]

0.5

1

Folic

aci

d up

take

(1

/fm

ol/m

g pr

otei

n/7

min

)

-Ki= 1.6 ± 0.2 µM)

0 10 20

1.5

-20 -10

Methotrexate [µM]

0.5

1

Folic

aci

d up

take

(1

/fm

ol/m

g pr

otei

n/7

min

)

-Ki= 1.6 ± 0.2 µM)

0.5

1

Folic

aci

d up

take

(1

/fm

ol/m

g pr

otei

n/7

min

)

-Ki= 1.6 ± 0.2 µM)

0 10 200 10 20

0 10 20

Folinic acid [µM]

0.5

1

Folic

aci

d up

take

(1/f

mol

/mg

prot

ein/

7 m

in)

-20 -10

-Ki = 2.1±0.3µM

0 10 20

Folinic acid [µM]

0.5

1

Folic

aci

d up

take

(1/f

mol

/mg

prot

ein/

7 m

in)

-20 -10

-Ki = 2.1±0.3µM

0.5

1

Folic

aci

d up

take

(1/f

mol

/mg

prot

ein/

7 m

in)

-20 -10

-Ki = 2.1±0.3µM

Page 44 of 51

45

Figure 6

0.5

1

1.5

3 [H

]-Fo

lic

acid

upt

ake

(1/f

mol

/mg

prot

ein/

7 m

in)

-1 -0.5 0 0.5 1

Sulfasalazine[mM]

-Ki =0.13±0.01 mM

0.5

1

1.5

3 [H

]-Fo

lic

acid

upt

ake

(1/f

mol

/mg

prot

ein/

7 m

in)

-1 -0.5 0 0.5 1

Sulfasalazine[mM]

-Ki =0.13±0.01 mM

Page 45 of 51

46

A

B

Figure 7 (contd.)

0

50

100

Contro

lfo

lt-1

RNAi

Who

le a

nim

al u

ptak

e (%

)

Contro

l

folt-

1RNAi

3[H]-Folic acid 14[C]-Ascorbic acid

0

50

100

Contro

lfo

lt-1

RNAi

Who

le a

nim

al u

ptak

e (%

)

Contro

l

folt-

1RNAi

3[H]-Folic acid 14[C]-Ascorbic acid14[C]-Ascorbic acid

0

3

6

Contro

lRel

ativ

e fo

lt-1

mR

NA

exp

ress

ion

over

β-a

ctin

(in

fol

ds)

folt-

1 RNAi

0

3

6

Contro

lRel

ativ

e fo

lt-1

mR

NA

exp

ress

ion

over

β-a

ctin

(in

fol

ds)

folt-

1 RNAi

Page 46 of 51

47

C

D

E

Figure 7

Mr folt-1-/- Wild-type

1 2 3 4 5 6 7 8 9

40,000

15,00010,0003,0542,0361,636

1,018

517

1040 bp

2250 bp

Mr folt-1-/- Wild-type

1 2 3 4 5 6 7 8 9

40,000

15,00010,0003,0542,0361,636

1,018

517

40,000

15,00010,0003,0542,0361,636

1,018

517

1040 bp1040 bp

2250 bp2250 bp

0

100

Wild

type

folt-

1-/-

KO

Wild

type

folt-

1-/- KO

Who

le a

nim

al u

ptak

e (%

)

3[H]-Folic acid

50

3[H]-Biotin

0

100

Wild

type

folt-

1-/-

KO

Wild

type

folt-

1-/- KO

Who

le a

nim

al u

ptak

e (%

)

3[H]-Folic acid

50

3[H]-Biotin

C. elegans genomic segment: V:11141304…11142870

11142 k

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5

folt-1 (C06H2.4)

ok1460

Deleted region

C. elegans genomic segment: V:11141304…11142870

11142 k

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5

folt-1 (C06H2.4)

ok1460

Deleted region

C. elegans genomic segment: V:11141304…11142870

11142 k

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5

folt-1 (C06H2.4)

ok1460

Deleted region

Page 47 of 51

48

Figure 8

Page 48 of 51

49

A

B

Figure 9

0

3000

6000

9000

Fluo

resc

ence

Int

ensi

ty

(in

pixe

ls)

0

Larva 1

Adult

Larva

2

Young

Adu

lt0

3000

6000

9000

Fluo

resc

ence

Int

ensi

ty

(in

pixe

ls)

0

Larva 1

Adult

Larva

2

Young

Adu

lt

0

2.5

5

Larva

1

Larva

2

Young

Adu

lt

Adult

Rel

ativ

e m

RN

A e

xpre

ssio

n (i

n fo

lds)

0

2.5

5

Larva

1

Larva

2

Young

Adu

lt

Adult

Rel

ativ

e m

RN

A e

xpre

ssio

n (i

n fo

lds)

0

2.5

5

Larva

1

Larva

2

Young

Adu

lt

Adult

Rel

ativ

e m

RN

A e

xpre

ssio

n (i

n fo

lds)

0

2.5

5

Larva

1

Larva

2

Young

Adu

lt

Adult

Rel

ativ

e m

RN

A e

xpre

ssio

n (i

n fo

lds)

Page 49 of 51

50

A

B

Figure 10

0

1

2

Folate

over-

supp

lemen

tedCon

trolR

elat

ive

folt

-1m

RN

A e

xpre

ssio

nov

er β

-act

in (

in f

olds

) 0

1

2

Folate

over-

supp

lemen

tedCon

trolR

elat

ive

folt

-1m

RN

A e

xpre

ssio

nov

er β

-act

in (

in f

olds

)

0

0.5

1.0Fo

late

over

-supp

lemen

ted

Contro

l

[3 H]

-fol

ic a

cid

upta

ke(f

mol

/5 a

nim

als/

5 m

in)

0

0.5

1.0Fo

late

over

-supp

lemen

ted

Contro

l

[3 H]

-fol

ic a

cid

upta

ke(f

mol

/5 a

nim

als/

5 m

in)

Page 50 of 51

51

A) Control

B) Over supplemented

C) GFP expression levels in the intestine

Figure 11

0

50

100

Contro

l

Fola

te

over

supp

lem

ente

d

GFP

flu

ores

cenc

e in

tens

ity

(%)

0

50

100

Contro

l

Fola

te

over

supp

lem

ente

d

GFP

flu

ores

cenc

e in

tens

ity

(%)

Page 51 of 51