(Received for publication, December 29, 1994)
From the
L1210 leukemia cells transport reduced folates and methotrexate
via a well defined reduced folate carrier system and, in the absence of
low folate selective pressure, do not express an alternate endocytotic
route mediated by cell surface folate receptors. This laboratory
previously described an L1210 leukemia cell line, MTXA,
with acquired resistance to methotrexate (MTX) due to the loss of
mobility of the reduced folate carrier. We now report on the
transfection of MTX
A with a cDNA encoding the murine
homolog of the human folate receptor isoform of KB cells to produce
MTX
A-TF1, which constitutively expresses high levels of
FR-
. MTX
A-TF1 and L1210 cells were utilized to compare
transport of methotrexate mediated by FR-
and the reduced folate
carrier, respectively. Methotrexate influx in the two lines was similar
when the extracellular level was 0.1 µM, but as the
methotrexate concentration increased, influx via the reduced folate
carrier increased in comparison to influx mediated by FR-
.
Transport kinetics indicated both a
20-fold lower influx K
and V
for
MTX
A-TF1 as compared to L1210 cells. The two cell lines
exhibited distinct influx properties. Methotrexate influx in
MTX
A-TF1 was markedly inhibited by 50 nM folic
acid and metabolic poisons. In L1210 cells, 1.0 µM folic
acid did not affect MTX influx, and metabolic poisons either had no
effect on or increased methotrexate influx. Removal of extracellular
chloride markedly inhibited transport in MTX
A-TF1 but
stimulated influx in L1210 cells. When the pH was decreased to 6.2,
methotrexate influx was not altered in MTX
A-TF1 but was
reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit
methotrexate influx in both L1210 and MTX
A-TF1 cell lines;
however, inhibition in MTX
A-TF1 could be accounted for on
the basis of inhibition of methotrexate binding to FR-
. The data
indicate that the reduced folate carrier and FR-
function
independently and exhibit distinct properties. FR-
expressed at
sufficient levels can mediate influx of MTX and folates into cells at
rates comparable to the reduced folate carrier and hence has
pharmacologic and physiologic importance.
In eukaryotic cells, import of a folate vitamin is required to
sustain enzymatic pathways leading to the synthesis of thymidine,
purines, and amino acids. The folate-dependent enzymes have long been
exploited as chemotherapeutic targets. For example, the folate analog
methotrexate (MTX) ()is a successful chemotherapeutic agent
due to its potent inhibition of dihydrofolate reductase (DHFR). Newer
antifolates that directly block folate-dependent purine and pyrimidine
biosynthesis (reviewed in (1) ) are now in clinical trials.
Membrane transport of antifolates such as MTX has been an area of
considerable interest because of the role that this process plays as a
determinant of the cytotoxicity, selectivity, and resistance to this
class of agents(2, 3, 4, 5) .
Folates and antifolates including MTX enter cells via two major
transport systems. The reduced folate carrier has received the most
attention because properties of antifolate transport mediated by this
system correlate well with the pharmacologic profile of these drugs (2, 3, 4) . This system has the
characteristics of carrier mediated
processes(6, 7, 8) , generates transmembrane
gradients(6, 9, 10) , and has a higher
affinity for MTX and reduced folates (1-5 µM)
than for folic acid (100-200
µM)(9, 11, 12, 13) . A
cDNA encoding this carrier, or a component of the carrier system, has
recently been cloned, and the sequence of the protein suggests that it
is a member of a superfamily of membrane-spanning
transporters(14, 15) .
The second transport system
mediates transport of folates by glycosylphosphatidylinositol-anchored
folate receptors (reviewed in (16, 17, 18) )
via an endocytotic process. Several folate receptor isoforms
(designated FR-, FR-
, and FR-
) have been cloned from
human and murine tissues in this and other
laboratories(19, 20, 21, 22, 23, 24, 25) .
The receptors have a very high affinity for folic acid (
1
nM) and the reduced folates 5-formyl- and
5-methyltetrahydrofolate (10-40 nM) and a lower affinity
for MTX and other 4-amino-antifolates (0.15-1.7 µM) (16, 17, 18) . Folate receptors are highly
expressed in some malignant tissues, suggesting a role in meeting
cellular folate requirements and as determinants of antifolate
cytotoxicity(26, 27, 28) . The physiologic
and pharmacologic properties of folate receptor-mediated transport have
not been characterized with the same degree of quantitation and depth
as for the reduced folate carrier.
In this report we characterize,
in detail, MTX transport mediated by FR-, the murine homolog of
the isoform expressed in KB cells(21) . To exclude any
contribution to transport via the reduced folate carrier, an L1210
leukemia line (MTX
A) was utilized in which the reduced
folate carrier is not functional(29) . This line was
transfected with a cDNA encoding murine FR-
and a clonal subline,
MTX
A-TF1, was isolated that constitutively overexpressed
this receptor. MTX was utilized as the transport substrate because upon
entering the cell it binds rapidly to DHFR, permitting very accurate
influx determinations over long intervals obviating back-flux
components that occur with natural
folates(6, 7, 9) .
The results of this
study indicate that transport mediated by FR- is fundamentally
different from transport mediated by the reduced folate carrier in
terms of energy, ion, and pH dependence. FR-
-mediated transport of
MTX into cells was quite rapid; MTX influx in the MTX
A-TF1
line was equivalent to influx mediated by the reduced folate carrier in
L1210 cells when the extracellular concentration was 0.1
µM. No evidence was found to support a linkage between
these two transport processes. Inhibition of FR-
-mediated
transport by agents that are inhibitors of transport via the reduced
folate carrier (i.e. BSP and probenecid) could be attributed
to inhibition of MTX binding to the folate receptor. The results of
this study are consistent with the concept that folate
receptor-mediated transport of folates and antifolates can achieve
influx rates comparable to those achieved by the reduced folate carrier
and can be a pathway of both physiologic and pharmacologic importance.
Figure 1:
Northern analyses of
L1210, MTX-resistant L1210 (MTXA), and MTX
A
transfected with FR-
(MTX
A-TF1). Upper panel,
total RNA (10 µg) from L1210 (lane1) and
MTX
A cells grown in complete RPMI 1640 medium (lane2) and from MTX
A-TF1 cells (TF1)
grown in either complete RPMI 1640 medium (lane3)
folate-free RPMI 1640 medium containing 0.5 nM folic acid (lane4) or folate-free RPMI 1640 medium containing
GAT (lane5) was hybridized with an oligonucleotide
probe specific for transfected FR-
. The blot was stripped and
hybridized with specific probes for DHFR and actin. Lower
panel, poly(A)
-selected RNA (5 µg) from
MTX
A, L1210, and MTX
A-TF1 was hybridized with a
probe specific for the reduced folate carrier
(RFC1).
The MTXA-TF1 transfectant
expresses a functional membrane FR-
at a level of 8.9 ± 0.6
nmol/g dry weight of cells (or 1.3 ± 0.1 pmol/10
cells) as assessed by [
H]folic acid binding
at 0 °C. Based on Scatchard analysis (Fig. 2), the K
for [
H]folic acid binding
was 1.9 ± 0.5 nM, in agreement with values for
endogenous murine and human FR-
(33, 36) . Over
the concentration range of 10 nM to 1 µM, no
specific binding of folic acid to L1210 or MTX
A could be
detected consistent with the lack of expression of FR-
in these
lines (Fig. 1).
Figure 2:
Specific binding of
[H]folic acid to L1210, MTX
A-TF1, and
MTX
A cells. Specific binding was measured at the indicated
concentrations of [
H]folic acid at 0 °C.
, L1210;
, MTX
A-TF1;
,
MTX
A. Inset, Scatchard analysis of the binding
data for MTX
A-TF1. The graph is representative of three
experiments.
Consistent with the high affinity of FR-
for folic acid(33, 36) , expression of FR-
in
MTX
A-TF1 resulted in a 400-fold lower folic acid growth
requirement compared to L1210 or MTX
A (Fig. 3, upper panel). The very small increase in the folic acid growth
requirement observed for MTX
A, which lacks a functional
reduced folate carrier, compared to L1210 cells demonstrates that the
reduced folate carrier is not a major uptake route for folic acid in
either cell line. Compared to MTX
A, both the
MTX
A-TF1 and L1210 cell lines exhibited a 35-fold lower
growth requirement for 5-formyltetrahydrofolate, a good substrate for
both FR-
(33, 36) and the reduced folate carrier (6, 9, 11, 30, 37) (Fig. 3, middle panel). Overexpression of FR-
at the level
achieved in the MTX
A-TF1 line appears to compensate
completely for the loss of the reduced folate carrier in meeting
cellular demands for folate, even at concentrations of
5-formyltetrahydrofolate comparable to physiologic
5-methyltetrahydrofolate levels.
Figure 3:
Folic acid (upper panel) and
5-formyltetrahydrofolate (middle panel) growth requirements
and inhibition of growth by MTX (lower panel). Cells were
plated at 8 10
cells/ml in the presence of the
indicated concentrations of folate or MTX and incubated for 72 h as
described under ``Materials and Methods.''
, L1210;
, MTX
A-TF1;
, MTX
A. Error bars are
S.E. of three experiments.
When grown in the presence of 2.3
µM folic acid (RPMI 1640 medium), the high level of MTX
resistance in the MTXA line is maintained in the
MTX
A-TF1 transfectant (Fig. 3, lower
panel). An extracellular folic acid concentration of 10 nM (a level which fully supports growth of MTX
A-TF1; Fig. 3, upper panel) resulted in only a small increase
in the sensitivity of the MTX
A-TF1 transfectant to MTX
(data not shown). These observations are consistent with transport
studies (see below) demonstrating that low levels of folic acid abolish
MTX uptake mediated by FR-
.
Figure 4:
Influx of [H]MTX in
L1210, MTX
A-TF1, and MTX
A cells. Cells were
pretreated with acid saline, washed, resuspended in HEPES-buffered
saline and, at time zero, exposed to 0.1 µM [
H]MTX at 37 °C.
, L1210;
, MTX
A-TF1;
, MTX
A cells. Error
bars are S.E. of 5 experiments.
The
[H]MTX associated with MTX
A-TF1 cells
was within the intracellular compartment and not simply bound to
FR-
on the cell membrane since an HBS acid wash (pH 4.5) following
[
H]MTX uptake removed only a trivial amount of
drug (data not shown).
The ratio of [H]MTX
influx in MTX
A-TF1 to L1210 cells decreased as the
extracellular MTX concentration was increased (Fig. 5),
suggesting that transport in MTX
A-TF1 cells saturates at a
lower MTX concentration with a lower influx V
than in L1210 cells. This was indeed the case. As shown in Table 1, the measured influx, K
, for MTX in
the MTX
A-TF1 transfectant (0.21 µM) is 1/20
that of L1210 cells (4.2 µM), and is consistent with the
MTX K
for FR-
(0.14
µM)(33) . The influx V
for
MTX in the MTX
A-TF1 line (0.18 nmol/g dry weight/min) is
1/16 that observed for L1210 cells (2.9 nmol/g dry weight/min). The
level of expression (MTX B
) of FR-
in
MTX
A-TF1 (8.9 nmol/gram dry weight) is 6 times that
observed for the reduced folate carrier in L1210 cells (1.5 nmol/g dry
weight)(29) . Hence the cycling rate for the reduced folate
carrier in L1210 cells is 100 times faster than the cycling rate for
FR-
in MTX
A-TF1. (
)In contrast, influx of
MTX in the MTX
A line was consistently less than 2% (1/75)
of influx in L1210 cells at extracellular MTX concentrations ranging
from 0.1 to 10 µM (Fig. 5). This is consistent with
a similar K
(3.9 µM) and greater than
90-fold lower V
for MTX influx in
MTX
A in comparison to L1210 cells (Table 1).
Figure 5:
The relationship between extracellular
[H]MTX concentration and the ratio of
[
H]MTX influx in MTX
A-TF1 or
MTX
A in comparison to L1210 cells. Cells were pretreated
with acid saline, washed, and resuspended in HEPES-buffered saline and,
at time zero, exposed to the indicated concentrations (0.1 µM to 10 µM) of [
H]MTX at 37
°C. Data points represent the ratio of influx in
MTX
A-TF1 or MTX
A in relation to influx in L1210
cells.
, MTX
A-TF1;
MTX
A cells.
Error bars are S.E. of two to four
experiments.
Figure 6:
The effects of folic acid on
[H]MTX influx in L1210, MTX
A-TF1, and
MTX
A cells. Upper panel, cells were pretreated
with acid saline, washed, resuspended in HEPES-buffered saline, and, at
time zero, exposed to 0.1 µM [
H]MTX
at 37 °C, in the presence (opensymbols) or
absence (closedsymbols) of 1.0 µM unlabeled folic acid.
,
= L1210;
,
= MTX
A-TF1.
=
MTX
A cells in the presence or absence of 1.0 µM folic acid. Error bars are S.E. of three to five experiments. Lower panel, effects of folic acid on 0.1 µM [
H]MTX influx. Data are expressed as influx
as a percentage of the level in the absence of folic acid.
,
L1210;
, MTX
A-TF1. Each point is the mean of two
experiments.
While structurally unrelated to folates, the
organic anion BSP is a potent inhibitor of the reduced folate carrier
and has been used to discriminate between folate uptake mediated by
this and other routes (35, 38, 39, 40) . BSP (150
µM) inhibited [H]MTX influx in L1210
and MTX
A-TF1 by 97% and 73%, respectively. Increasing the
BSP concentration to 350 µM completely abolished the
residual [
H]MTX flux in the MTX
A-TF1
line (Table 2). Probenecid is also an inhibitor of MTX transport
by the reduced folate
carrier(41, 42, 43, 44) . At a
concentration of 1 mM, probenecid markedly inhibits
[
H]MTX influx in L1210 cells but has only a small
effect on influx in MTX
A-TF1. However, at a level of 10
mM, probenecid markedly suppresses
[
H]MTX influx in both cell lines (Table 2).
Despite structural dissimilarity, the antibiotic CI-920 also competes
with reduced folates for the reduced folate carrier(45) . At a
level of 150 µM, CI920 markedly suppresses
[
H]MTX influx in L1210 cells while inhibiting
influx in the MTX
A-TF1 line by less than 10% (Table 2). In the presence or absence of folic acid, BSP,
probenecid, or CI-920 inhibition of MTX influx in the MTX
A
line was negligible (less than 2% of control rate in L1210 and
MTX
A-TF1; Fig. 4Fig. 5Fig. 6and data
not shown).
As shown in Fig. 7, each of the reduced folate
carrier inhibitors also inhibit MTX binding to FR-. BSP and
probenecid inhibit MTX binding with IC
values of 100
µM and 4 mM, respectively. The IC
for CI-920 was not quantitated but is somewhat less than
probenecid. In general, for all of the agents tested, there is good
correlation between inhibition of [
H]MTX influx
and binding to FR-
in MTX
A-TF1 (Table 2). Hence,
the effects of these agents on [
H]MTX influx can
be attributed to inhibition of [
H]MTX binding to
FR-
. Folic acid is a highly specific inhibitor of FR-
, and
CI-920 is the most selective inhibitor of the reduced folate carrier.
MTX binding to the reduced folate carrier was below the level of
detection in this assay.
Figure 7:
Effects of BSP, probenecid, or CI-920 on
[H]MTX binding to FR-
. Specific binding of
0.1 µM [
H]MTX was measured in
MTX
A-TF1 cells in the presence of the indicated
concentrations of BSP, probenecid, or CI-920 at 0 °C.
, BSP;
, probenecid;
, CI-920. The graph is representative of
three experiments. There is no detectable specific binding of MTX to
L1210 or MTX
A cells under these
conditions.
The major circulating folate,
5-methyltetrahydrofolate, has been utilized extensively for the
characterization of folate uptake in MA-104
cells(38, 46, 47) . Since
5-methyltetrahydrofolate has a 20-fold higher affinity for FR-
then does MTX(33, 36) , it was anticipated that its
transport would be less sensitive to organic anions and this was indeed
the case. However, inhibition of the binding and influx of
[
H]5-methyltetrahydrofolate by either probenecid
or BSP was comparable (Table 2). Folic acid, an even stronger
ligand for the FR-
(150-fold lower K
compared
to MTX) is insensitive to the effects of BSP on binding and transport
in MTX
A-TF1. There is no measurable transport of 50 nM folic acid into L1210 cells under these conditions.
Energy,
Temperature, pH, and Ion Dependence of
[H]MTX
Influx-MTX
A-TF1 and L1210 cells were used to
compare various physical, chemical, and metabolic perturbations on
[
H]MTX influx mediated by FR-
or the reduced
folate carrier (Table 3). The energetics of MTX transport in
L1210 cells is unusual(8, 10, 48) . An
energy-requiring efflux pump results in enhanced net cellular transport
of MTX in the presence of metabolic inhibitors. Metabolic poisons
either enhance or have no effect on influx(10, 48) .
[
H]MTX influx in L1210 cells was enhanced by 10
mM azide and unchanged by 0.5 mM dinitrophenol (DNP).
In contrast, [
H]MTX influx in the
MTX
A-TF1 line was abolished by this concentration of azide
and markedly inhibited by dinitrophenol (Table 3). Hence MTX
influx in MTX
A-TF1 is highly energy-requiring and ceases
rapidly when energy metabolism is blocked.
The two cell lines also
had a markedly different pH dependence. [H]MTX
influx in L1210 cells was reduced by 70% when the pH of the transport
buffer was decreased from 7.4 to 6.2. [
H]MTX
influx in the MTX
A-TF1 transfectant was insensitive to this
change in pH. Influx of [
H]MTX in both lines is
temperature-sensitive. However, the Q
for MTX
A-TF1 cells (7.1) is twice that of L1210 cells
(3.8).
MTX influx via the reduced folate carrier is
sodium-independent, but is highly sensitive to the anionic composition
of the extracellular compartment and is inhibited by a variety of
inorganic and organic anions(9, 49, 50) .
Replacement of NaCl isosmotically with HEPES markedly stimulated influx
of [H]MTX in L1210 cells. However, under
identical conditions [
H]MTX influx in the
MTX
A-TF1 transfectant was reduced by 97% (Table 3).
MTX influx was not sensitive to the intra- or extra-cellular
Na
concentration in L1210 or MTX
A-TF1
cells. Hence influx was unchanged by substituting Na
with Li
and neither cell line showed sensitivity
to ouabain (Table 3). The residual MTX influx in MTX
A
was not significantly altered by azide, dinitrophenol, ouabain, or the
substitution of NaCl with HEPES (data not shown).
Stable expression of murine FR- in a cell line in which
the reduced folate carrier is present but immobile provided the
opportunity to explore, in detail, the influx characteristics of
transport mediated by FR-
. MTX was used as the probe for these
studies because of its unique qualities, which facilitate influx
measurements(6, 7, 9) .
The data provide
strong evidence that MTX influx in the MTXA-TF1 cell line
is mediated exclusively by FR-
and demonstrates that the folate
receptor can function independently of the reduced folate carrier. A
similar conclusion was made by Dixon et al.(51) utilizing a MTX-resistant human breast cancer cell
line (MTX
ZR-75-1)(52, 53) .
This
study defines several parameters that distinguish between
FR--mediated influx and influx mediated exclusively by the reduced
folate carrier. These two transport processes are fundamentally
different in many respects. 1) There is a marked difference in energy
dependence. Consistent with previous
reports(9, 10, 48) , influx of MTX in L1210
cells is impervious to the immediate effects of energy depletion.
However, influx of MTX in MTX
A-TF1 cells is markedly
inhibited by metabolic poisons, indicating a direct coupling of
FR-
-mediated transport to energy metabolism. 2) MTX influx in
L1210 cells is stimulated by the removal of chloride anion, a
manifestation of the nonspecific heteroexchange between anions and the
reduced folate
carrier(8, 9, 49, 54) . However,
this perturbation resulted in cessation of MTX influx in
MTX
A-TF1 cells. Although inorganic anions may have
generalized effects on receptor-mediated endocytosis(55) ,
these data are consistent with previously reported chloride enhancement
of folate binding to rat kidney FR(56) . Initial studies
suggest a decrease in the MTX binding affinity for FR-
in the
absence of chloride (data not shown). 3) MTX influx in L1210 cells is
markedly suppressed when the pH is decreased from 7.4 to 6.2. However,
over this pH range, there is no change in MTX transport in
MTX
A-TF1 cells. This is consistent with reports that
binding of folates to the folate receptor does not diminish until the
pH falls below 5.0(57) . The data indicate that a proposed
alternative route for MTX transport, optimized at pH 6.2, cannot
account for rapid influx in MTX
A-TF1(35) . 4) While
MTX influx in both cell lines is temperature-dependent, the influx
Q
in MTX
A-TF1 is 2-fold greater. 5) Finally,
MTX influx in MTX
A-TF1 was 5 orders of magnitude more
sensitive to folic acid than was L1210 cells. Folic acid is a poor
substrate for the reduced folate carrier (K
>200
µM)(6, 9, 11, 12) , but
has a very high affinity for FR-
(33, 36) .
FR-
expression in MTX
A-TF1 allowed for growth in low
levels of folic acid and 5-formyltetrahydrofolate even in the absence
of a functional reduced folate carrier, consistent with similar reports
by other
laboratories(51, 58, 59, 60, 61) .
Since FR-
has a 20-fold higher binding affinity for
5-formyltetrahydrofolate compared to
FR-
(33, 36) , the specific folate receptor
isoform expressed in normal and malignant cells may influence the
efficacy and therapeutic index of treatment regimens that utilize this
folate. MTX resistance is maintained in MTX
A-TF1 even in
the presence of very low levels (10 nM) of folic acid, a
consequence of the potent inhibitory effect of folic acid on MTX influx
in this cell line. Thus, the dual effects of low folate on folate
receptor bearing cells in vitro (maintaining cell growth and
at the same time blocking FR-mediated MTX cytotoxicity) indicates that
folate receptor-mediated transport could support the growth of a
reduced folate carrier-defective tumor cell without increasing that
cells sensitivity to MTX.
Transport mediated by FR- was quite
rapid; the rate of influx of 0.1 µM MTX in
MTX
A-TF1 cells and L1210 cells was identical. However, as
the extracellular MTX level was increased, influx mediated by the
reduced folate carrier dominated (Fig. 5), the result of both a
20-fold lower influx K
and V
in MTX
A-TF1 as compared to L1210 cells. When the
relative expression of each transporter was considered along with the
influx V
, the cycling rate of the reduced folate
carrier in L1210 cells (116 molecules MTX/binding site/h) was
calculated to be 2 orders of magnitude greater than that of the
FR-
-mediated process in MTX
A-TF1 (1.2 molecules of
MTX/binding site/h). The cycling rate for FR-
in the present study
is comparable to rates reported for FR-
expressed in L1210 and
MA-104 cells (0.8-1.1 molecules/binding site/h) (39, 46) and is more than adequate to meet the modest
folate requirement of cells under physiologic conditions (16) .
Hence when expressed to a sufficient level, FR-
may be a
significant transport route for folates at physiologic concentrations
(10-50 nM) and for antifolates at low blood levels.
However, FR-
becomes a very minor contributor (Fig. 5) to
transport at higher (pharmacologic) levels of folates and antifolates,
when the reduced folate carrier dominates.
Folate receptor isoforms have distinct binding affinities for folates and antifolates(33, 36) . The rate of transport at a given folate or antifolate concentration will therefore depend not only on the amount of receptor present but on the specific isoform expressed. Recent studies have shown that folate receptors are widely expressed in normal and malignant tissues and that the pattern of expression is isoform-specific with striking overexpression in certain carcinomas (26, 27, 28) . Specificity in transport mediated by different folate receptor isoforms may be an important element in the development of tumor-specific chemotherapeutic regimens.
While it is clear that folate transport in MTXA-TF1
cells is dependent upon the initial association of drug with folate
receptor, the remaining steps in the transport process are not clear
but probably involve endocytosis of the receptor-ligand
complex(62) . In MA104 cells, a monkey kidney epithelial cell
line, FR-
, as well as other glycosylphosphatidylinositolanchored
proteins, appears to cluster in non-clathrin-coated invaginations or
caveolae(46, 47, 63, 64) . However,
there is recent evidence that this process may not be limited to
caveolae(65) . It has been suggested that following
acidification of the sealed-off caveolar vesicle, freed folate ligand
enters the cytoplasm via the reduced folate carrier. This proposal was
based on the observation that the process is sensitive to
probenecid(66) . However, the present study indicates that the
effects of probenecid and BSP on MTX and 5-methyltetrahydrofolate
influx in MTX
A-TF1 can be explained on the basis of
inhibition of binding to FR-
. These agents, therefore, cannot
reliably discriminate between carrier- and folate receptor-mediated
influx. Hence, probenecid sensitivity does not imply participation of
the reduced folate carrier in folate influx in MTX
A-TF1
cells. These observations, together with the multiple qualitative
differences in transport noted above, support the conclusion that the
reduced folate carrier and FR-
operate independently.