From the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada
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ABSTRACT |
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The membrane topology of the human reduced folate
carrier protein (591 amino acids) was assessed by single insertions of
the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of
these constructs were probed with anti-hemagglutinin epitope monoclonal
antibodies to assess whether the insertion was exposed to the external
environment or to the cytoplasm. The results are consistent with the
12-transmembrane topology predicted for this protein. The hemagglutinin
epitope insertion mutants were also tested for their effects on the
function of the reduced folate carrier. For these studies, each of the
constructs had a carboxyl-terminal fusion of the enhanced green
fluorescent protein to monitor and quantitate expression. Insertions
into the external loop between transmembrane regions 7 and 8 (Pro-297),
the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225),
and near the cytoplasmic amino and carboxyl termini (Pro-20 and
Gly-492, respectively) had minor effects on methotrexate binding and
uptake. The insertion into the cytoplasmic loop between transmembrane
regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of
methotrexate, whereas the insertion into the external loop between
transmembrane regions 11 and 12 (Pro-427) selectively interfered with
uptake but not binding.
Mammalian cells require reduced folates for several biochemical
pathways involving purine, pyrimidine, and amino acid metabolism. Since
mammals cannot synthesize folates, these compounds must be obtained in
the diet and imported by cells via either the
glycosylphosphatidylinositol-linked folate receptor (1-5) or the
reduced folate carrier
(RFC),1 an integral membrane
protein (6-9). A third, low pH-dependent component for
folate transport has been reported (10, 11), but its involvement in
folate delivery is not clearly understood. The RFC is the main route
for transport of 5-methyltetrahydrofolate, the major circulating folate
in the bloodstream, 5-formyltetrahydrofolate (folinic acid), and the
folate analog, methotrexate (Mtx) (9, 12-14).
The rfc gene has been cloned from hamster (15), human
(16-19), murine (20), and
rat2 sources. The identity of
the rfc gene was verified by cDNA transfections that
restored Mtx sensitivity to RFC-deficient cells (15-20). The human RFC
protein is 591 amino acids as deduced from its cDNA sequence, and
the hydropathy profile predicts 12 transmembrane-spanning (TM) domains
with the amino and carboxyl termini located in the cytoplasm. Recently,
the cytoplasmic location of the carboxyl-terminal tail has been
confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails
than the human RFC (15-20), although all three RFCs share about 68%
identical or similar amino acid residues. The human RFC is glycosylated
at an asparagine residue in the first extracellular loop (9, 22, 23),
and while the hamster RFC contains the conserved sequence in this loop
(15), its glycosylation status has not been confirmed. The murine RFC
lacks the conserved glycosylation site and is not glycosylated
(20).
The deduced 12-TM topology can place the RFC in a large family of major
transport facilitators that includes the bacterial Lac permeases and
the mammalian glucose transporters (24, 25). The lack of an ATP-binding
domain in the RFC suggests that it does not belong to the 12-TM family
of ABC transporters that includes the multi-drug resistance protein
(P-glycoprotein), and the cystic fibrosis transmembrane conductance
regulator (26). The three-dimensional structure is known for only a
small number of membrane proteins, although for many others the
structures have been deduced by statistical and biophysical models (27,
28). The modeled structure or topology for each protein must be
verified before it is possible to understand how the extracellular,
intracellular, and TM domains contribute to the function of the protein.
The topology of a number of proteins with transmembrane domains has
been elucidated by glycosylation scanning mutagenesis (29, 30),
truncation and fusion strategies (31-33), and by epitope insertion
into putative loops (34-40). In this study, we have inserted the
hemagglutinin epitope (HA) (41, 42) of the human influenza virus into
nine sites of the human RFC. These HA insertion constructs were
transfected into a Chinese hamster ovary (CHO) cell line defective for
RFC expression. The accessibility of the epitope to anti-HA monoclonal
antibodies was evaluated in permeabilized and non-permeabilized cells
to determine its orientation. The results support the predicted 12-TM
structure of the RFC. In addition, clones expressing these insertion
mutants have been evaluated for Mtx binding and transport.
Chemicals--
Restriction endonucleases and modifying enzymes
were obtained from Amersham Pharmacia Biotech, except for
CpoI (MBI Fermentas). Unlabeled Mtx was obtained from Sigma;
[(3',5',7-3H]Mtx was purchased from Moravek Biochemicals
(Brea, CA);
N5-formyltetrahydrofolate (folinic acid) was purchased
from ICN Biomedicals, and Polybrene was from Aldrich. Geneticin®
(G418-Sulfate), LipofectAMINETM and Opti-MEM® I were
purchased from Life Technologies, Inc.
DNA Sequencing--
Correct insertion of the HA epitopes was
initially confirmed by DNA sequencing using the T7 polymerase kit from
Amersham Pharmacia Biotech and using [ Cells and Cell Culture--
Wild-type Pro
For transient transfections, MtxRII 5-3 cells were grown on sterile
round glass coverslips placed in 24-well tissue culture plates and
incubated with 0.2 µg of plasmid DNA complexed with LipofectAMINETM. After addition of
The phenotype of transfectants selected for growth in low folinic acid
medium were tested for Mtx resistance by dose-response curves, as
described previously (43). The G418-selected transfectant clones were
used for Mtx uptake and binding assays (46, 47). In all experiments, at
least two independently selected clones were used for the analyses.
Construction of HA Insertion Mutants--
The human influenza
virus HA epitope (YPYDVPDYA) was inserted into selected sites of the
rfc cDNA by two strategies. In the first approach, based
on that described by Canfield and Levenson (34), the cDNA was
digested at unique restriction endonuclease sites (ApaI,
254; NotI, 883; CpoI, 1374; StuI,
1570; numbering based on the human rfc cDNA reported in
Ref. 16) predicted to be in intra- or extracellular loops. These were
blunt-ended using either nuclease S1 or the Klenow fragment of DNA
polymerase I, as appropriate to maintain reading frame, and treated
with calf intestinal alkaline phosphatase. HA oligonucleotides of the
sense strand were synthesized with either one or two additional G
deoxyribonucleotides at the 5' end (see oligos 1 and 2 of Table
I). Each was annealed with an equimolar
amount of the antisense HA oligonucleotide (oligo 3) which contains two
additional G residues at the 5' end. The double-stranded products were
blunt-ended either with nuclease S1 or the Klenow fragment of DNA
polymerase I, as appropriate to maintain reading frame, treated with T4
polynucleotide kinase, and ligated using molar ratios of
insert:linearized plasmid of 5:1 and 1:1. The HA insertion constructs
were used to transform XL1-Blue Escherichia coli
(Stratagene). Positive clones were selected by hybridization with
[
The second HA insertion strategy was based on the two-step PCR method
described by Howard et al. (35), using the primer sets and
templates listed in Table II.
The PCR reactions contained standard PCR buffer (48), 1 ng of template,
and optimized levels of MgCl2. In some cases the reactions
included 10% Me2SO. The PCR cycler parameters were as
follows: (i) "hot start": 1 min at 94 °C, then 5 min at
80 °C, during which the Taq DNA polymerase was added;
(ii) 26 cycles of amplification: 1 min at 94 °C, 1 min at 52 °C,
2.5 min at 72 °C; and (iii) final extension for 8 min at 72 °C.
The products were separated on agarose gels, and the band of the
correct size was purified by the Geneclean procedure (Bio 101). The two
"first-step" products for each set were diluted, mixed, and used as
the template for the second round of PCR. The extension primers for
each reaction were the same as used in the first round of PCR (Table
II), and the same PCR conditions were used. The second-step products
were purified as above, digested with either HindIII and
EcoRI or ApaI and EcoRI, and cloned
into the p5' Construction of p5'
The HA insertion constructs in the p5' Immunofluorescence Labeling and Microscopy--
The
transiently transfected cells on coverslips were washed twice
with PBS (1× PBS, pH 7.4, contains 137 mM NaCl,
2.7 mM KCl, 8.3 mM
Na2HPO4, 1.5 mM
KH2PO4, 0.9 mM
CaCl2, and 0.5 mM MgCl2) and fixed
30 min at room temperature with freshly prepared 2% paraformaldehyde in 0.5× PBS. The cells were washed twice with PBS
containing 1% bovine serum albumin and 0.02% sodium azide (PBS-plus).
Cells in one well of each series were permeabilized with 0.2% Triton
X-100® in PBS at room temperature and then washed three times with
PBS-plus. All cells were incubated at room temperature with 12CA5 mouse
monoclonal antibody (Roche Molecular Biochemicals) and then washed
twice with PBS-plus. Permeabilized and non-permeabilized cells were
washed once with PBS containing 0.05% Triton X-100 to remove
nonspecifically bound antibody. The cells were washed twice with
PBS-plus and stored overnight at 4 °C in PBS-plus. The next day, the
cells were incubated at room temperature with goat anti-mouse IgG,
fluorescein isothiocyanate conjugate (BIOSOURCE International, Camarillo, CA) in PBS-plus containing 10% goat serum.
The cells were washed as before. The coverslips were placed on slides
with mounting medium containing the DNA binding fluor DAPI, sealed, and
viewed with a Zeiss Axioskop fluorescence microscope with 488 nm
excitation. Images were captured using a Sony CCD digital camera and
analyzed using Northern EclipseTM (version 2.0)
software (Empix Imaging, Inc., Toronto, Ontario, Canada).
Mtx Uptake and Binding--
Uptake and binding experiments were
performed as described previously (46, 47) using EGFP-tagged clones of
the HA insertion mutants selected for G418 resistance and green
fluorescence at the plasma membrane. Total cellular protein was
quantified by the Bradford method (49). To normalize for specific
expression of the HA-RFC-EGFP fusion proteins, the mean fluorescence
values for each clone was determined by flow cytometry (FACScan,
Becton-Dickinson) using MtxRII 5-3 cells as the control for cell size
and background fluorescence parameters. The clones expressing the
RFC-EGFP fusion proteins had mean fluorescence values at least 15 times
greater than the control cells.
Western Blotting--
G418-selected HA-RFC-EGFP transfectant
cells were pelleted after growth in suspension culture, and ~2 × 106 cells were washed twice with chilled PBS,
resuspended in 100 µl of protease protection buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% SDS,
1.0% Nonidet P-40, 0.5% sodium deoxycholate, 60 µg/ml
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 20 µg/ml pepstatin, 0.2 mM orthovanadate, and 5× concentrated Protease inhibitor mixture tablets (Roche Molecular Biochemicals)), and flash frozen at 5' Epitope Insertion Analysis of RFC Topology--
The epitope
insertion approach was chosen for examining the membrane topology of
RFC because it also allows an assessment of the functional consequences
of disrupting the chosen insertion site. The nine insertion sites
chosen in the RFC (Fig. 1) were the
largest inner or outer loops predicted by the TMpred algorithm (27) as
well as the amino- and carboxyl-terminal tails. In addition, these
regions contain less than 50% amino acid conservation among the
hamster, mouse, and human homologues.
The HA insertion constructs (lacking EGFP) and control plasmids were
transiently transfected into MtxRII 5-3 cells. The accessibility of the
HA epitope to antibody binding was compared in permeabilized and
non-permeabilized cells (Fig. 2). If the
RFC is properly localized to the plasma membrane, one would expect to
see green fluorescence at the periphery of the cells expressing each
construct, similar to the localization reported recently for a
carboxyl-terminal HA fusion of the human RFC (22). This plasma membrane
localization was seen in most of the transfectants analyzed in this
study. In the cells expressing the constructs HA-P20, HA-V152, HA-S225, HA-Q385, or HA-G492, the HA epitope is clearly facing the cytoplasm since fluorescence at the membrane surface is only detectable in the
permeabilized cells. For the HA-Q385 construct, the fluorescence was
mainly membrane-localized but punctate in nature. In the case of
HA-V152, few cells showed fluorescence. In the cells transfected with
the HA-R263 construct, epitope detection required permeabilization. In
this case, few cells were detected in which the fluorescence was
located at the cell membrane, and unlike most of the other transfectants, this was distributed in a punctate manner. This could be
due to inaccessibility of the epitope to the anti-HA antibodies, lack
of proper expression of the protein, or to protein degradation (see
below). The HA epitope in constructs HA-G54, HA-P297, and HA-P427 is on
the external face of the membrane, since it is accessible to anti-HA
antibody in both non-permeabilized and permeabilized cells. In the
cells expressing HA-P427, fluorescence was detected in the
non-permeabilized case but was somewhat diffuse and punctate in nature
with a majority of cells showing a membrane localization of the
fluorescence. Permeabilization of the cells resulted in a clearer
membrane localization of the fluorescence which was still somewhat
punctate. The reasons for this are not clear but may represent a
reduced accessibility of the antibodies to the epitope. These epitope
orientations are consistent with the predicted protein topology
presented in Fig. 1.
Transient transfections can result in large variations in expression
and can affect protein localization, leading to intracellular inclusions (51, 52). To ensure that the determined HA epitope orientations were a true reflection of RFC topology, the experiment was
repeated using stable clones of cells selected in low folinic acid
medium and expressing either the HA-P20, HA-G54, HA-S225, HA-P297, or
HA-G492 constructs. A similar pattern of localization was found for the
stable clones, although there was a stronger, more uniform detection at
the plasma membrane and fewer intracellular inclusions than their
transient counterparts (data not shown).
To ensure that the HA-tagged constructs were properly localized,
transient transfections were also carried out with the HA-RFC-EFGP fusions to permit an analysis independent of antibody detection. In
general, the EGFP moiety had little or no discernible influence on the
cells expressing constructs HA-P20, HA-V152, HA-S225, HA-P297, HA-Q385,
HA-P427, or HA-G492 since the fluorescence patterns obtained from the
HA detection and the respective EGFP fusions of each construct (without
antibody detection) had very similar distributions (Fig. 2).
Transfections with either the HA-G54-EGFP or HA-R263-EGFP constructs
yielded very few fluorescent cells.
Transfectants bearing these nine HA-RFC-EGFP constructs were selected
in G418, thus using a selection criterion independent of RFC
functionality. In the initial transfections, 20-40% of the
G418-resistant colonies showed fluorescence at the plasma membrane.
This enabled screening for expressing cells that were subsequently
cloned. As expected, most clones showed a predominant plasma membrane
localization of fluorescence except for those expressing HA-G54-EGFP,
HA-R263-EGFP, or HA-V152-EGFP. In the former two, the cells showed
diffuse fluorescence throughout the cytoplasm and nucleus, whereas in
the latter one, a majority of the cells contained diffuse fluorescent
material with a minority fluorescing primarily at the plasma membrane
(Fig. 3, panel C). The diffuse
fluorescent pattern is similar to the distribution observed with the
soluble EGFP control (Fig. 2, panel A).
The large perinuclear inclusions in cells expressing either
HA-P297-EGFP (Fig. 3, panel F) or HA-P427-EGFP (Fig. 3,
panels H and I) appear to result from trapping in
the Golgi as determined by use of a medial Golgi-specific antibody
(MG-160, data not shown). In addition, there are also many smaller
inclusions throughout the cytoplasm of these cells (Fig. 3,
panel J).
Analysis of RFC Function--
To examine the effect of the epitope
insertions on RFC function, the transfected cell lines were evaluated
for their ability to grow in low folinic acid, to take up
[3H]Mtx, and to bind [3H]Mtx.
The insertion of the HA epitope at four sites (HA-P20, HA-S225,
HA-P297, and HA-G492) had little discernible effect on the ability of
RFC to rescue MtxRII 5-3 cells under low folinic acid conditions (Table
III), whereas the efficiency of colony formation with the HA-G54
construct was reduced 5-6-fold. Clones of HA-P20, HA-G54, HA-S225,
HA-P297, and HA-G492 selected under low folinic conditions were assayed
for sensitivity to Mtx. All clones, except those expressing the HA-G54
construct, had wild-type sensitivity to the drug (results not shown).
The HA-G54 stable transfectants showed Mtx sensitivity that was
intermediate between the wild-type and the MtxRII 5-3 response (not
shown). None of these transfected constructs were tested for binding or
uptake of folates since previous work has shown that cells able to grow
in low levels of folinic acid can bind and take up folates (15, 16,
45-47).
MtxRII 5-3 cells transfected with either the HA-V152, HA-R263, HA-Q385,
or HA-P427 constructs were unable to grow in low folinic acid medium,
suggesting that the insertions at these sites affect RFC function. It
is of interest to note that in transient transfections only a small
number of cells transfected with HA-R263 have the protein located at
the cell membrane (see above).
To examine the ability of cells expressing the nine constructs to take
up Mtx, G418-selected isolates were used. Stable clones of
5'
Representative clones expressing either the HA-P20-EGFP, HA-S225-EGFP,
HA-P297-EGFP, or HA-G492-EGFP constructs, in which the fusion protein
is localized to the membrane, were capable of taking up Mtx (Table
IV). This is consistent with the Mtx
sensitivity and growth in low folinic acid medium demonstrated for
these transfected constructs without the EGFP fusion. The normalized
Mtx uptake in cells expressing either HA-S225-EGFP or HA-P297-EGFP is
about 70 and 45%, respectively, relative to the control, whereas those expressing HA-P20-EGFP and HA-G492 were 80 and 120%, respectively, of
the control. Western blotting with anti-GFP (Fig.
4) showed that each of these
transfectants produces a broad band (probably the result of
glycosylation) around 105 kDa and comparable to the 5'
Cells expressing either the constructs HA-G54-EGFP, HA-V152-EGFP,
HA-R263-EGFP, HA-Q385-EGFP, or HA-P427-EGFP failed to transport Mtx.
This inability to take up Mtx may be due to functional inactivation of
the fusion protein that is otherwise properly targeted and inserted in
the plasma membrane, degradation of the fusion proteins, improper
localization, or improper processing of the protein. Cells expressing
either HA-G54-EGFP, HA-V152-EGFP, or HA-R263-EGFP have little, if any,
of the fluorescent fusion protein localized to the plasma membrane
(Fig. 3). This probably accounts for the inability of these constructs
to restore RFC function to the RFC-deficient cells. Western blots
probed with anti-GFP antibody show that cells expressing HA-G54-EGFP
have a discrete band of about 45 kDa (Fig. 4, lane 6). The
fluorescence in these cells is indicative of the complete translation
of the fusion protein. However, the degradation product is larger than
the soluble EGFP protein (~27 kDa, Fig. 4, lanes 1 and
14), suggesting that a portion of the amino terminus of the
RFC has been removed. The HA-V152-EGFP product is not evident on the
blot presented (Fig. 4, lane 7), but longer exposures show a
faint broad band corresponding to ~105 kDa. This is probably the
material that is localized to the plasma membrane in a small fraction
of the cells expressing this construct. Bands of lower molecular size
that might represent the material responsible for the diffuse
fluorescence were not detected in longer exposures of the Western blot.
Because only a small portion of cells expressing this construct have
the product localized to the plasma membrane, it is not yet possible to
examine the effect of this HA insertion on RFC function. The
HA-R263-EGFP cell extract (Fig. 4, lane 9) has small amounts
of two degradation products with apparent masses of ~45 and ~25 kDa
which are visible only in longer exposures of the Western blot (not shown).
The cells expressing either the HA-Q385-EGFP or the HA-P427-EGFP
constructs have strong fluorescence at their plasma membranes although
the HA-P427-EGFP-expressing cells also have some intracellular material. This indicates, for the most part, that the fusion proteins are correctly localized and maintained under G418 selection (Fig. 3,
panels G and H). The Western blot pattern for the
non-functional HA-Q385-EGFP resembles that of the functional
5'
The Mtx binding capabilities of the G418-selected HA-RFC-EGFP clones
(or a FACS-enriched population) were assayed, and representative data
for six of the constructs are presented in Fig.
5. Since each clone expresses a different
amount of the HA-RFC-EGFP fusion protein, the binding data were first
normalized to the total amount of cellular protein and then to the mean
fluorescence determined by flow cytometry. The clones expressing either
HA-P20-EGFP or HA-G492-EGFP transport Mtx (Table IV) and have
normalized binding values about 80% that of the control cells (Fig.
5). The HA-S225-EGFP and the HA-P297-EGFP expressing clones which have
about 70 and 45%, respectively, of the uptake (Table IV) also have
about half the Mtx binding of the control cells (Fig. 5). This suggests
that the lowered Mtx uptake may be a direct consequence of reduced binding.
The clones expressing either HA-Q385-EGFP or HA-P427-EGFP do not
transport Mtx and have different phenotypes with respect to binding.
The cells expressing the HA-Q385-EFGP construct present the HA epitope
on the cytoplasmic side of the plasma membrane and bind Mtx only 20%
as well as the control cells. Conversely, the HA-P427-EGFP-transfected
cells, with the HA insertion in the last extracellular loop, bind 40%
as much Mtx as the control cells. However, cells expressing
HA-P427-EGFP have about half the fluorescent material at the membrane
(see Fig. 3, panels H and I) where initial substrate binding would be expected to take place. Thus, the binding capability of this clone may be similar to that of the control transfectants since the normalization procedure did not take into account the cellular localization of the fusion protein. This suggests
that the insertion in the last extracellular loop does not affect Mtx
binding but rather implicates it in the substrate translocation process.
A summary of the results of the functional analysis of cells expressing
the HA-RFC-EGFP constructs is shown in Table
V.
In this report, the topology of the human RFC was assessed by an
epitope insertion technique that also allows partial analysis of the
functional regions of the protein. Although the RFC had not been
previously subjected to the same level of systematic topology
determination that has been applied to other transmembrane proteins
(34-40), previous reports suggested that the loops between TMs 1 and 2 (9, 23) and between TMs 5 and 6 (53) are facing the cell exterior and
that the carboxyl terminus is located in the cytoplasm (22). The
results presented here confirm these findings and localize other RFC
regions. The amino terminus, the loops between TMs 4 and 5, 6 and 7, and 10 and 11 all face the cytoplasm, whereas the loop between TMs 11 and 12 faces the cell exterior.
Although the results of the HA epitope orientation analysis are not
exhaustive since every single putative loop has not been targeted, they
are consistent with the 12-TM topology predicted for the RFC (Fig. 1).
The confirmation of this topology is not an insignificant goal. Placing
the RFC within a group of major transport facilitators (24, 25) that
includes the Lac permease of E. coli, the mammalian glucose
transporters, and many other proteins, may help elucidate functional
domains by comparison with other family members. All these proteins
have a 12-M topology in which the amino and carboxyl termini are
intracellular, and the patterns of loop sizes are strongly conserved,
although the amino acid sequences are not. For example, each member of
the 12-M protein family mentioned above has a very large inner loop between TMs 6 and 7, which for cystic fibrosis transmembrane
conductance regulator and P-glycoprotein contains the nucleotide
binding domain and, in others, has an undefined function. The
carboxyl-terminal tail is the most variable region with respect to
length, even among the four species for which the RFC has been
characterized, although its functional role is unclear.
In this study, the EGFP tag was used to monitor construct expression in
living cells during the clonal selection process in G418 medium. In
addition, the EGFP marker allowed us to quantify the level of protein
expressed from the constructs and to assess its localization and
degradation status.
The addition of the EGFP fusion at the carboxyl terminus of the
wild-type RFC was shown not to affect the ability of the protein to
bind or take up Mtx. The green fluorescent protein has been used in
many studies of protein trafficking and localization within the cell
since it does not influence the targeting of the protein to which it is
fused (52, 54-58). Cells transfected with the HA insertion constructs
HA-P20, HA-S225, HA-P297, and HA-G492 had colony formation efficiencies
in low folinic acid medium similar to those transfected with the
wild-type RFC, regardless of whether they were fused to the EGFP.
However, the EGFP tag did reduce further the colony forming efficiency
of cells transfected with the HA-G54 construct which, when transfected
by itself under low folinic acid growth conditions, yielded about
15-20% as many colonies as the wild-type RFC. The HA-G54 insertion
does not disrupt the glycosylation site but is in a region suspected to
be critical for substrate recognition/discrimination as single amino
acid substitutions in the corresponding loop of the murine RFC affect substrate binding (21, 59). The cells expressing the HA-G54 construct
may therefore have a reduced ability to bind or transport reduced
folates, although this was not further explored.
The insertion of the HA epitope at four sites resulted in loss of
function independent of the EGFP fusion. For two of these sites
(Val-152 and Arg-263) the loss of function could be attributed to a
lack of stable localization of the protein at the plasma membrane.
Thus, we cannot discern the roles of these two sites in Mtx binding and
uptake. Clones expressing the HA-V152-EGFP construct showed
fluorescence at the plasma membrane which, with extended passage in
G418 medium, became diffuse throughout the cytoplasm although no fusion
protein degradation products were detected in Western blots. Cells
expressing the HA-R263-EGFP construct showed a diffuse pattern of
fluorescence in the cytoplasm and nucleus. The nonfunctional status of
the HA-R263-EGFP protein may be a result of disrupted secondary
structure, improper localization, or impaired function leading to
degradation. The boundaries of the TM regions vary when a polytopic
membrane protein is analyzed by multiple prediction programs or
biophysical models (28). Thus, the HA-R263 insertion, predicted to be
close to the membrane, may interrupt the transmembrane region.
Alternatively, the hydrophobic HA epitope at this location may result
in a novel transmembrane helix. Nonetheless, the other insertion
(HA-S225) in this predicted loop indicates that at least a nearby
region faces the cytoplasm.
Cells expressing HA-Q385-EGFP have the fusion protein localized to the
plasma membrane, but they bind Mtx very poorly. Because this insertion
site is in a cytoplasmic loop and should not interact directly with the
substrate, the loss of binding is likely due to structural changes in
the RFC.
The cells expressing the HA-P427-EGFP construct appear to have about
half the Mtx-binding ability of the control, but there is evidence that
the insertion at this site interferes with the substrate translocation
process and not binding (see Table IV, Fig. 5). The Mtx binding of the
cells expressing HA-P427-EGFP is comparable to that of cells expressing
either the HA-P297-EGFP or HA-S225-EGFP constructs, both of which
transport Mtx. Furthermore, although binding was normalized to the
total amount of cellular protein and mean fluorescence, the precise
amount of fluorescent material that is at the plasma membrane was not
considered. No attempt was made to measure accurately the amount of the
fusion protein as techniques for purifying membrane proteins generally aim for product purity rather than quantitative recovery. As well, there is no assurance that all of the protein localized to the membrane
is functional. For cells expressing either the HA-P297-EGFP or the
HA-P427-EGFP construct, it was evident from microscopy that about half
of the fluorescent material was not at the plasma membrane. This was
corroborated by Western blots showing that about half of the detected
material was not of the size expected for the RFC-EGFP fusion protein.
With this data taken into account, the normalized Mtx binding of the
HA-P427-EGFP construct would be similar to that of the control.
However, unimpaired Mtx binding by insertion at Pro-427 does not rule
out the involvement of this loop in substrate binding as it is possible
that the insertion would not disrupt a site contributed by several
external loops.
Of the four constructs competent for Mtx uptake, HA-P297-EGFP has the
potential to interfere directly with the substrate interaction since
the insertion is in an external loop. Cells expressing this construct
have about half of the fusion protein at the cell membrane and about
50% of the Mtx binding and uptake relative to the control. Thus, at
this level of analysis, there is no apparent effect on substrate
interaction. A single amino acid change in this loop in the murine
RFC-1 was recently shown to result in changes in Mtx uptake via a
4-fold increase in Km values without affecting
Vmax (53).
The insertions in the intracellular regions corresponding to the
amino-terminal (HA-P20-EGFP) and the carboxyl-terminal regions (HA-G492-EGFP) had no obvious effects on Mtx uptake or binding. Cells
expressing HA-S225-EGFP showed a roughly parallel reduction in Mtx
binding and uptake compared with the transfectants expressing the
5' Not every loop of the RFC was targeted for HA insertion. Some are very
short and may serve no purpose other than providing a hinge for the
next helix to insert in the membrane. Thus, insertion in these loops is
more likely to inactivate the protein by disrupting structure rather
than selectively destroying a binding site or substrate translocation region.
The work described here is a step toward understanding the structure of
the RFC protein. The identification of the exterior and cytoplasmic
surfaces of this protein will guide future work characterizing the
sites involved in Mtx binding and translocation through the membrane
and possible regulatory interactions with other proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) obtained from ICN Biomedicals. The entire sequence of each
construct was verified by the ABI 377 sequencing facility in the
Robarts Research Institute (London, Ontario, Canada).
3 and
Pro
3 MtxRII 5-3 CHO cells (MtxRII 5-3) were maintained as
monolayer cultures in
-medium supplemented with 10% fetal bovine
serum as described previously (43). MtxRII 5-3 is an Mtx-resistant cell
line that is defective in reduced folate transport because it does not
express the rfc mRNA (15).
-medium containing
20% fetal bovine serum to each well, the cells were incubated a
further 18 h prior to immunofluorescence labeling and microscopy.
Stable transfectants of MtxRII 5-3 cells were obtained using the
Polybrene procedure described previously (44). Stable clones of the HA
insertion constructs in either the 5'
RFC or 5'
RFC-EGFP background
(described below) were selected and maintained in folic acid-free
medium containing 10% dialyzed fetal bovine serum and 2 nM
folinic acid (45). Stable clones of MtxRII 5-3 cells transfected with
the 5'
RFC-EGFP fusion products were also selected with G418 (1.2 mg/ml) in
-medium supplemented with 10% fetal bovine serum.
Individual colonies resistant to G418 were screened by fluorescence
microscopy to ensure expression of the fusion protein. Three to five
clones of each transfectant from the low folinic acid and the G418
selection regimes were isolated by limiting dilution from independently generated transfectants.
-32P]dCTP end-labeled oligo 3 and verified by DNA
sequencing. Because of the insertion strategy at the CpoI
site (HA-P427), an extra valine residue was inserted at the carboxyl
end of the HA epitope.
Oligonucleotides for constructing HA insertions
RFC and p5'
RFC-EGFP vectors described below. The HA
insertion mutants are named according to the amino acid that precedes
the HA epitope (i.e. HA-P20 is the HA epitope inserted after
Pro-20).
Oligonucleotides for HA insertions by two-step PCR
RFC and p5'
RFC-EGFP--
The expression
plasmid pRFC contains nt
94 to +1937 of the rfc cDNA
from pHuMtxT4 (16) cloned into the pcDNA3 expression vector
(Invitrogen). This construct lacks most of the rfc 3'-UTR including the polyadenylation signal. Plasmid p5'
RFC contains nt +1
to +1937 of the human RFC. The enhanced green fluorescent protein (EGFP) was fused in-frame to the carboxyl terminus of the
rfc gene in p5'
RFC by replacing the stop codon with a
BglII site. Using oligos 4 and 5 (Table I) as PCR primers,
the EcoRI-BglII fragment of the rfc
(nt 1697-1870) was amplified and cloned into a pcDNA3-based
plasmid which contained the BamHI-NotI fragment of pEGFP-N1 (CLONTECH). The resulting plasmid,
containing the 3' end of the rfc gene fused to the EGFP, was
linearized with HindIII and EcoRI, and the
HindIII-EcoRI fragment of p5'
RFC was inserted
to produce p5'
RFC-EGFP.
RFC and p5'
RFC-EGFP
backgrounds were purified by the Qiagen Plasmid Midi Kit (Qiagen, Chatsworth, CA). The EGFP fusion constructs were used in transfections to determine the cellular localization of the protein and for the Mtx
binding and uptake studies; the HA insertion constructs without the
fusion were used for anti-HA immunofluorescence microscopy to determine
protein topology.
80 °C. After the samples were
thawed on ice, the proteins were precipitated with 10 volumes of
acetone, resuspended in Laemmli buffer (50), and separated on SDS-PAGE
gels. An extract equivalent to ~5 × 105 cells was
loaded per lane. The proteins were transferred to nitrocellulose membranes, and the RFC-EGFP fusion proteins were detected by the ECL
procedure (Amersham Pharmacia Biotech) using polyclonal anti-GFP (CLONTECH) and horseradish peroxidase-labeled
anti-rabbit secondary antibody (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RFC-EGFP Fusion Proteins--
Before examining the topology
of the RFC in transfected cells using the HA epitope, the EGFP tag was
fused in-frame to the carboxyl terminus of various constructs
containing the wild-type RFC. This was done to determine whether the
EGFP moiety affected RFC function, as it was desirable to have a tag to
monitor protein expression and cellular location. MtxRII 5-3 cells were
transfected with plasmid DNA from either RFC, RFC-EGFP, 5'
RFC, or
5'
RFC-EGFP constructs and selected in low folinic acid medium (45).
There was no significant difference in numbers of colonies obtained for
these four constructs, indicating that neither the 94 base pairs of the
rfc 5'-UTR nor the EGFP fusion affect the ability of the
transfected RFC to complement functionally the RFC-deficient cell line
(Table III). Colonies selected from these
transfections exhibited wild-type sensitivity to Mtx, as expected (data
not shown). In a second set of experiments (not shown), kinetic
analysis of [3H]Mtx uptake was carried out to determine
the apparent Kt and Vmax
values (46). For these experiments, stable clones that expressed either
the human RFC, the RFC-EGFP fusion, or 5'
RFC were selected for
growth in low levels of folinic acid. The cells transfected with the
RFC construct had an apparent Kt of 2.0 ± 0.6 µM and Vmax of 3.3 ± 1.2 pmol/min/mg total protein for transporting Mtx. The cells expressing
the RFC-EGFP fusion protein had similar kinetic parameters with an
apparent Kt of 1.2 ± 0.7 µM and
Vmax of 5.0 ± 1.0 pmol/min/mg total
protein. The values for cells expressing 5'
RFC were
Kt of 1.7 ± 0.7 µM and
Vmax of 5.0 ± 1.2 pmol/min/mg total
protein. Based on these results neither the removal of the 5'-UTR nor
the carboxyl-terminal EGFP fusion affected the functionality of RFC,
confirming the suitability of this tag as a passive reporter. The EGFP
moiety can then be used to monitor the expression of the RFC, since
fluorescence should only occur if the fusion protein were properly
translated. In addition, since the fusion protein was shown to be
functional, it must be properly localized, and thus the fluorescence
could serve as an indicator of the cellular location of the fusion
protein (see below).
Transfection frequencies for phenotypic complementation
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Fig. 1.
Proposed topological structure of the human
RFC. This topology is based on the program TMpred (27). No
secondary structure is implied for the loops. The longer transmembrane
helices in the shaded region are drawn perpendicular to the
membrane surface for clarity, although these helices may actually be
shorter or inserted into the membrane at an angle. The HA insertion
sites are shown by filled arrowheads. The HA insertion
mutant nomenclature indicates the amino acid that precedes the HA
epitope. 1, HA-P20; 2, HA-G54; 3,
HA-V152; 4, HA-S225; 5, HA-R263; 6,
HA-P297; 7, HA-Q385; 8, HA-P427; 9,
HA-G492. Sites 2, 5, 8, and 9 are at unique
restriction endonuclease sites in the RFC cDNA coding region. The
HA epitope was inserted into sites 1, 3, 4, 6, and
7 by a two-step PCR process (see "Experimental
Procedures").
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Fig. 2.
Fluorescence microscopy of cells transiently
expressing the HA epitope constructs. HA epitope orientations in
each construct were assessed in non-permeabilized (left
column) and permeabilized (middle column) cells using
the murine anti-HA monoclonal antibody 12CA5. The secondary antibody is
fluorescein-conjugated anti-mouse IgG. The right column
shows the EGFP fusion constructs. In most of the panels in the
left column (panels D, G, J, M, P, V, and
BB), as well as in panels B and R, the
image captures of DAPI-stained nuclei are overlaid on the image
captures of the green fluorescence emission to confirm the presence of
cells. The transfected constructs in each panel are as follows:
A, soluble EGFP; B, 5' RFC control for
nonspecific antibody binding; C, 5'
RFC-EGFP; D
and E, HA-P20; F, HA-P20-EGFP; G and
H, HA-G54; I, HA-G54-EGFP; J and
K, HA-V152; L, HA-V152-EGFP; M and
N, HA-S225-EGFP; O, HA-S225-EGFP; P
and Q, HA-R263; R, HA-R263-EGFP; S and
T, HA-P297; U, HA-P297-EGFP; V and
W, HA-Q385; X, HA-Q385-EGFP; Y and
Z, HA-P427; AA, HA-P427-EGFP; BB and
CC, HA-G492; DD, HA-G492-EGFP. All images were
captured at the same magnification. Scale bar (panel
DD), 20 µm.
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Fig. 3.
Fluorescence microscopy of transfectants
selected in G418 and expressing the HA insertions in the RFC-EGFP
background. Panel A, HA-P20-EGFP; B,
HA-G54-EGFP; C, HA-V152-EGFP, arrow points to
plasma membrane distribution; D, HA-S225-EGFP; E,
HA-R263-EGFP; F, HA-P297-EGFP; G, HA-Q385-EGFP;
H, HA-P427-EGFP; I and J, enlarged
view of cells expressing HA-P427-EGFP; K, HA-G492-EGFP;
L, 5' RFC-EGFP; M, overlay of MtxRII 5-3 cells
after image captures for DAPI staining and green fluorescence. The
image in panel I was focused on the membrane-localized
material and the large perinuclear inclusion (arrow). The
same cells in panel J focused on the small cytoplasmic
granules. The scale bar for panels I and
J (shown in J) is 20 µm. All other panels to
same scale, represented by scale bar in panel M, 20 µm.
RFC-EGFP, HA-P20-EGFP, HA-S225-EGFP, HA-P297-EGFP, HA-Q385-EGFP, HA-P427-EGFP, and HA-G492-EGFP were obtained and further analyzed. The
clones of HA-G54-EGFP, HA-V152-EGFP, and HA-R263-EGFP were not stable
and gradually stopped producing the fusion protein. Clones expressing
these three constructs were sorted by FACS to enrich for the
RFC-EGFP-expressing cells prior to use in the experiments below.
RFC-EGFP
control (Fig. 4, lane 4). The non-glycosylated size is
predicted to be 91 kDa. The HA-P297-EGFP expressing clones, in which
the fusion protein is localized to both the plasma membrane and to
intracellular inclusions (Fig. 4, lane 10), also show some degraded product. This may correspond to the material in the
intracellular inclusions (not shown, but similar to the inclusions
shown for HA-P427-EGFP in Fig. 3, panels I and
J), although this was not further explored in the present
study. However, this suggests that the reported Mtx uptake value for
the clone expressing HA-P297-EGFP may be underestimated as the
normalization procedure did not take into account the cellular location
of the fusion protein (see "Discussion").
[3H]Mtx uptake in MtxRII 5-3 cells transfected with
HA-RFC-EGFP constructs
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Fig. 4.
Western blot of total proteins from the
G418-selected transfectants. The RFC-EGFP fusion products were
probed with anti-GFP and detected by chemiluminescence. Lanes
1 and 14, soluble EGFP controls; lane 2,
MtxRII 5-3; lane 3, wild-type control; lane 4, 5' RFC-EGFP; lane 5, HA-P20-EGFP; lane 6,
HA-G54-EGFP; lane 7, HA-V152-EGFP; lane 8,
HA-S225-EGFP; lane 9, HA-R263-EGFP; lane 10,
HA-P297-EGFP; lane 11, HA-Q385-EGFP; lane 12,
HA-P427-EGFP; lane 13, HA-G492-EGFP. Molecular mass
standards (kDa) are indicated at the left.
RFC-EGFP control (Fig. 4, lane 4). Cells expressing
HA-P427-EGFP also produce protein of the expected size, but about half
of the material migrates as smaller products of ~45 kDa (Fig. 4,
lane 12), which may correspond to the material in the small
intracellular inclusions (Fig. 3, panel J), although this
was not examined further in this study. Although the HA-P427-EGFP
construct has an extra valine at the carboxyl terminus of the HA
epitope (see "Experimental Procedures"), this is likely not a
contributing factor to disruption of function. This residue is
chemically similar to the hydrophobic nature of the HA epitope, and
alternative insertion strategies have included single copies with one
to four additional amino acids at the carboxyl terminus, or tandem, or
triplet copies of the HA epitope to boost antibody detection, with no
apparent detrimental effects on protein function (37, 39, 40). The
cells expressing either the HA-Q385-EGFP or HA-P427-EGFP constructs are
of particular interest because they produce protein of the expected
size that is localized to the plasma membrane, but they do not
transport Mtx (Table IV). This inability may be due to disruption of
the substrate-binding site(s) or of the substrate translocation
process. Binding assays were carried out to distinguish between these
two possibilities.
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Fig. 5.
Relative [3H]Mtx binding for
representative clones expressing HA-RFC-EGFP. Clones containing
constructs HA-G54-EGFP, HA-V152-EGFP, and HA-R263-EGFP are not shown
since they did not have the fusion protein localized to the plasma
membrane. The binding data are reported relative to the 5' RFC-EGFP
positive control cells that bound 300 pmol of Mtx per mg of total
cellular protein. The mutant MtxRII 5-3 cells bound 10 pmol per mg of
total protein (not shown).
Summary of functional testing of transfected HA-RFC-EGFP constructs
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RFC-EGFP control. These cells have most of the fusion protein localized to the plasma membrane, and the Western blot indicates that
it is of the appropriate size. This suggests that the effects of the
epitope inserted at this intracellular site may result from a subtle
disruption of RFC structure.
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ACKNOWLEDGEMENTS |
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We thank F. M. R. Williams and H. Sadlish for helpful comments during the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council of Canada (to W. F. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Biochemistry, University of Western
Ontario, London, Ontario N6A 5C1, Canada.
§ To whom correspondence should be addressed. Tel.: 519-661-3438; Fax: 519-661-3499; E-mail: flintoff{at}julian.uwo.ca.
2 S. A. Rubin, GenBankTM accession number U38180.
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ABBREVIATIONS |
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The abbreviations used are: RFC, reduced folate carrier; Mtx, methotrexate; CHO, Chinese hamster ovary; nt, nucleotide(s); HA, hemagglutinin epitope; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; PMSF, phenylmethylsulfonyl fluoride; TM, transmembrane-spanning; oligo, oligonucleotide.
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REFERENCES |
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