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J. Biol. Chem., Vol. 277, Issue 36, 33325-33333, September 6, 2002
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From the Departments of
Received for publication, June 14, 2002
The major pathway for cellular uptake of
the water-soluble vitamin folic acid in mammalian cells is via a plasma
membrane protein known as the reduced folate carrier (RFC). The
molecular determinants that dictate plasma membrane expression of RFC
as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to
image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the
polypeptide and not within the cytoplasmic NH2- or
COOH-terminal domains of the protein. Further, the integrity of the
hRFC backbone is critical for export of the polypeptide from the
endoplasmic reticulum to the cell surface. This trafficking is
critically dependent on intact microtubules because microtubule
disruption inhibits motility of hRFC-containing vesicles as well as
final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.
Folate is necessary for the synthesis of precursors of nucleic
acids, initiation of protein synthesis in mitochondria, and metabolism
of certain amino acids (1, 2). Deficiency of this essential
micronutrient leads to a variety of abnormalities including derangement
of one-carbon metabolism and inhibition of growth. Humans and other
mammals do not possess the ability to synthesize folate and must obtain
this vitamin from exogenous sources by intestinal absorption.
Absorption occurs from both dietary sources in the small intestine and
from bacterially synthesized folate in the large intestine (3, 4).
Thereafter, folate is distributed into different body compartments
where it is taken up by individual cell types for use in different
metabolic reactions. Many previous studies have shown that the main
folate uptake pathway in cells occurs via a specialized
carrier-mediated mechanism, the reduced folate carrier
(RFC1; in humans, known as
hRFC) (4-6). The RFC is also involved in intestinal absorption of
folate from the small and large intestine (5). The functional
properties of this transport pathway have been well characterized
(4-6) and its molecular identity established after cloning of its
cDNA (7-9) and characterization of its flanking regulatory regions
(4).
In contrast, comparatively little is known about the mechanisms that
control the intracellular trafficking of the RFC protein and its
appropriate expression in the plasma membrane, topics of particular
importance for transport of folate by mammalian epithelial cells.
Genetic defects in such folate transport processes have been reported
previously; however, little is known about the exact sites of these
defects (10). To address these questions, we used high resolution
confocal imaging techniques to monitor the distribution and transport
of hRFC fusion proteins tagged with the enhanced green fluorescent
protein (EGFP) in living mammalian epithelial cell lines. By comparing
the expression patterns of nine truncated hRFC mutants, we show that
targeting of hRFC to the plasma membrane depends on the integrity of
the membrane-spanning "backbone" of the protein, rather than on
targeting sequences in the NH2- or COOH-terminal
cytoplasmic regions. Furthermore, by using video-rate confocal
microscopy, we resolved the intracellular trafficking dynamics of
hRFC-containing vesicles and demonstrate that delivery of hRFC-EGFP to
the cell surface is critically dependent on intact microtubules but not
microfilaments. Taken together, these results highlight how the hRFC
polypeptide sequence is targeted and delivered to the cell surface
within a physiologically relevant context, and they define the regions
of the hRFC protein in which mutations would impair folate transport by
disrupting trafficking and targeting mechanisms.
Materials--
FM4-64 was from Molecular Probes (Eugene, OR).
Green (EGFP-N3) and yellow (EYFP-membrane) fluorescent
protein vectors were from BD Biosciences (Palo Alto, CA). Cytochalasin
D, nocodazole, colchicine, and Construction of hRFC-EGFP and Truncated Constructs--
cDNA
encoding the open reading frame of the full-length hRFC and
various truncated constructs (Fig. 1)
were generated by PCR using the primer sequences and combinations shown
in Table I. PCRs were carried out as
described previously (11), and the resulting products were gel
isolated and ligated together with the EGFP coding sequence digested
from the EGFP-N3 vector. This method generated in-frame fusion proteins
under the control of the human cytomegalovirus promoter. The nucleotide
sequence of each resulting construct was confirmed by sequencing
(SeqWright, Houston, TX and Laragen, Inc., CA).
Cell Culture and Transient Transfection--
Human embryonic
kidney (HEK-293) cells, human duodenal derived intestinal epithelial
(HuTu-80) cells, and Madin-Darby canine kidney cells were maintained in
minimum Eagle's medium containing 10% fetal bovine serum. Human colon
adenocarcinoma (Caco-2) cells were maintained in Dulbecco's modified
Eagle's medium (20% fetal bovine serum) and human colonic (NCM 460)
cells in Ham's F-12 medium (20% fetal bovine serum). All media
contained glutamine, sodium bicarbonate, penicillin, and streptomycin.
For transient transfection, cells were grown on
poly-D-lysine-coated cover slips cemented in holes at the
bottom of sterile Petri dishes (MatTek, MA) and were transfected at
~90% confluence with plasmid DNA complexes using LipofectAMINE 2000 (Invitrogen). For all transfection protocols, conditions were optimized
to minimize the amount of DNA used (typically ~0.6 µg of cDNA
of the indicated construct and less than recommended by the
manufacturers) to minimize any nonspecific effects of fusion protein
overexpression. Monolayers were subsequently imaged using confocal
microscopy at indicated times after transfection (typically ~48 h).
Confocal Imaging of hRFC Constructs--
Cell monolayers were
monitored for hRFC-EGFP expression using a custom built laser scanning
confocal microscope (12) or a video-rate scanning confocal microscope
(13). Both instruments were based on Olympus IX70 inverted microscopes
fitted with 40×, NA 1.35 oil immersion objectives. EGFP, EYFP, DsRed,
and FM4-64 were all excited using the 488 nm line from an argon ion
laser, and emitted fluorescence was monitored using a 530 ± 20 nm
band pass filter (EGFP, EYFP) or 650LP filter (DsRed, FM4-64). For slow
frame scanning, confocal images were obtained by scanning either
laterally (top view; x-y scans) or axially (side view; x-z scans) across the specimen.
For video-rate imaging experiments, images were recorded at 30 Hz (one
frame every 33 ms) from confocal sections with the microscope focused
about 2 µm above the coverglass within cells maintained at 22 or
37 °C. Data were digitized using the stream acquisition function of
the Metamorph processing package (Universal Imaging, Downingtown, PA).
The resulting image stacks (x-y time) were then
smoothed using a two-frame average to yield a final image stack at
66-ms frame intervals. The motions of individual vesicles were tracked
using the point-to-point tracking function in Metamorph, and the
resulting paths were exported to the Origin graphing package for
analysis. QuickTime videos of image sequences are appended as
supplementary material.
Flow Cytometry--
HEK-293 cells were used for these
experiments because their high (~40%) efficiency of transfection
allowed counting of large cell populations (15,000 cells for each
construct, three experiments). Monolayers of HEK-293 cells were
transfected in situ within individual T75 tissue culture
flasks using ~4 µg of cDNA encoding individual constructs.
Cells were trypsinized and resuspended in Ca2+-free
phosphate-buffered saline, and populations of >15,000 transfected cells were measured for intensity of fluorescence emission (488 nm
excitation, 530 ± 15 nm emission) using a FACStation fluorescence analysis system (Becton Dickinson Immunocytometry Systems).
Cellular Distribution of hRFC-EGFP--
To visualize the targeting
of hRFC in mammalian cells, we transiently transfected a variety of
epithelial cell lines with cDNA encoding the full-length fusion
construct (hRFC-EGFP) and analyzed the resulting fluorescence
distribution by confocal microscopy. In HuTu-80 cells (human duodenal
epithelium), hRFC-EGFP expression was evident at the cell surface and
in cellular processes extending from the cell membrane as well in a
population of discrete, intracellular vesicular structures (Fig.
2A, see also later). This
distribution of fluorescence contrasted markedly with HuTu-80 cells
expressing EGFP alone, in which the entire cytosolic volume was
fluorescent (Fig. 2B). These contrasting distributions were
also evident in axial (x-z) sections of cells expressing
hRFC-EGFP (Fig. 2C) or EGFP alone (Fig. 2D),
which suggested that hRFC-EGFP was targeted to all plasma membrane
domains of the cell. The localization of hRFC-EGFP to the plasma
membrane was not unique to HuTu-80 cells because a similar targeting
was evident from lateral (x-y) sections images in a variety
of other mammalian epithelial cell lines including HEK-293, Caco-2,
Madin-Darby canine kidney, and NCM-460 cells (Fig. 2E).
Identification of Polypeptide Domains Important for hRFC Expression
and Targeting--
To determine the domains within the hRFC protein
which are important for its correct trafficking and targeting we
compared the cellular distribution seen with the full-length hRFC-EGFP with that observed with the series of hRFC mutants diagrammed in Fig.
1.
Fig. 3 shows representative lateral
confocal images of HuTu-80 cells transiently transfected with each of
the indicated constructs and imaged 48 h later when the
full-length hRFC-EGFP was almost completely distributed to the plasma
membrane (see below). It is apparent that changes in the sequence of
hRFC resulted in a wide variety of cellular distributions. Four classes
of distribution were evident: 1) expression primarily at the cell
surface (hRFC[1-530]-EGFP, hRFC[1-452]-EGFP, and
hRFC[19-466]-EGFP, Fig. 3A); 2) cytosolic, non-membrane-bound expression (hRFC[452-591]-EGFP and
hRFC[1-27]-EGFP, Fig. 3B); 3) expression confined within
intracellular membranes (hRFC[1-301]-EGFP and hRFC[1-144]-EGFP,
Fig. 3C); 4) a heterogeneous distribution, spanning several
of the above categories (hRFC[28-591]-EGFP and
hRFC[302-452]-EGFP, Fig. 3D). Expression of
hRFC[28-591]-EGFP was evident at the cell surface as well as
throughout the intracellular compartments, whereas
hRFC[302-452]-EGFP was restricted to intracellular membranes soon
after transfection and thereafter appeared in the cytoplasm. This
latter profile is suggestive of a poorly tolerated protein structure
that is actively targeted for degradation (14).
To quantify the relative amounts of hRFC protein expressed in the
plasma membrane versus that in other cellular compartments, we imaged transfected cells that were costained by extracellular application of the red emitting lipophilic dye FM4-64 (15). This
selectively labels the plasma membrane, thereby allowing us to estimate
the extent of localization of hRFC-EGFP at the cell surface by
measuring the degree of overlap of red and green fluorescence. Examples
are shown in Fig. 3E for cells expressing hRFC-EGFP at the
cell surface (high degree of overlap with FM4-64: yellow
color) and cells expressing a construct (hRFC[1-301]-EGFP) that
remained localized to intracellular membranes (little fluorescence colocalization).
Fig. 3F shows measurements of fluorescence
colocalization obtained in this way for the various constructs. A high
degree (>50%) of colocalization was seen in cells expressing
hRFC-EGFP, hRFC[1-530]-EGFP, hRFC[1-452]-EGFP, and
hRFC[19-466]-EGFP, confirming that these four constructs targeted
efficiently to the cell surface. Little colocalization (~8-14%,
equivalent to that (~10%) with EGFP alone) was seen with
hRFC[1-301]-EGFP, hRFC[1-144]-EGFP, hRFC[1-27]-EGFP, hRFC[452-591]-EGFP, and hRFC[302-452]-EGFP, confirming their
inability to target to the cell surface. An intermediate degree of
colocalization (~ 28%) was seen with hRFC[28-591]-EGFP,
suggesting that a small proportion of the expressed construct was
delivered to the cell surface.
In addition to the differing cellular localization of hRFC-EGFP
proteins expressed by the different constructs, it was also apparent
that the total amount of protein expressed varied greatly among
constructs. For example, although hRFC-EGFP and hRFC[1-452]-EGFP were both expressed selectively at the cell surface, cells expressing the latter construct were much dimmer (at equivalent laser power) than
hRFC-EGFP-transfected cells. Given identical transfection conditions
(0.6 µg of DNA, imaged after 48 h) and promoter expression, this
variability presumably reflects differences in cellular processing for
each construct (e.g. relative rates of protein synthesis and degradation). To quantify these differences, we used flow cytometry to
measure the distributions of total fluorescence of cells transfected with constructs that showed predominant targeting to the plasma membrane (hRFC-EGFP, hRFC[1-530]-EGFP, hRFC[1-452]-EGFP,
hRFC[19-466]-EGFP) or to intracellular membranes
(hRFC[1-301]-EGFP and hRFC[1-144]-EGFP). HEK-293 cells were used
for these experiments because their high transfection efficiency
permitted measurements from large (>15,000) populations of cells. We
note however, that the subcellular distribution of each hRFC construct
was identical in both HEK-293 and HuTu-80 cell lines (Fig.
2C and data not shown).
Fig. 4 shows that although the level of
protein expression was not reduced appreciably by either partial
truncation of the COOH-terminal cytoplasmic tail (mean fluorescence of
the hRFC[1-530]-EGFP population 102 ± 3% of that of
hRFC-EGFP, three independent runs) or by partial truncation of both the
COOH-terminal and NH2-terminal regions
(hRFC[19-466]-EGFP, 84 ± 14%), complete truncation of the
COOH-terminal hRFC-EGFP sequence substantially decreased the level of
expression (hRFC[1-452]-EGFP, 42.7 ± 3%). Further truncations into the hRFC backbone reduced expression further (hRFC[1-301]-EGFP, 27.4 ± 3% and hRFC[1-144]-EGFP, 17.5 ± 3%). Taken
together, the data of Figs. 3 and 4 show that the hRFC structure
dictates the subcellular targeting as well as the efficiency of protein
expression within mammalian cells.
Trafficking of hRFC-EGFP Involves Microtubule-based
Transport--
In contrast to other membrane transporters (16-18),
little is known about the cytoskeletal mechanisms that direct the
intracellular trafficking of hRFC (11). To resolve this issue,
we examined the possible roles of microfilaments and microtubules in
transporting hRFC-EGFP to the cell surface by using pharmacological
methods to disrupt the cytoskeleton selectively.
To identify an appropriate time window for pharmacological
intervention, we began by characterizing the kinetics of hRFC-EGFP expression after transfection. Fluorescence was initially apparent after 4 h as a weak juxtanuclear signal. Subsequently, the
fluorescence increased, spread throughout the cell, and became
increasingly localized to the plasma membrane (Fig.
5A). The relative extent of
hRFC-EGFP expression at the cell surface was estimated by costaining with FM4-64, as before. At early times after transfection (~ 4-5 h)
there was little colocalization between hRFC-EGFP distribution and
FM4-64 staining (12.1 ± 2.5%, Fig. 5, B
left and C), and this value was similar to that
with EGFP alone (16.7 ± 6%; Fig. 5C). However, by
36 h colocalization between hRFC-EGFP and FM4-64 had increased to
a maximal value (63.5 ± 6%, Fig. 5, B
right and C) as a result of trafficking of
hRFC-EGFP to the cell surface, whereas colocalization in cells
transfected with EGFP alone (9.1 ± 6%) or hRFC[1-304]-EGFP
(13.8 ± 9%,
As shown in Fig. 5C, hRFC-EGFP fluorescence at the cell
surface began to increase rapidly about 12 h after transfection,
and we thus selected this as a suitable time point at which to apply cytoskeletal disrupting drugs, either cytochalasin D, a microfilament disrupting agent, or nocodazole, a microtubule-disrupting agent. Both
drugs were added to the incubation medium at final concentrations (400 nM) that are sufficient to disrupt cytoskeletal
architecture but minimize toxicity during the prolonged (8 h)
application (11, 20). Colocalization of FM4-64 and hRFC-EGFP
fluorescence was then assessed 8 h later (i.e. 20 h after transfection). Measurements are shown in Fig. 5D,
expressed as the change in colocalization relative to that measured at
12 h to reflect trafficking of hRFC-EGFP to the plasma membrane
during the time the drugs were present. Although colocalization of
fluorescence between hRFC-EGFP and FM4-64 increased to a similar extent
in untreated cells (2.2 ± 0.4-fold, n = 12 cells)
and cells treated with cytochalasin D (2.0 ± 0.3-fold,
n = 21 cells), a slight decrease was seen after incubation with nocodazole (0.91 ± 0.42-fold, n = 39 cells). Thus, disruption of the microtubular cytoskeleton almost
completely inhibited hRFC-EGFP trafficking to the plasma membrane,
whereas disruption of the actin cytoskeleton was without effect.
Transport Dynamics of hRFC-containing Vesicles--
Confocal
images of hRFC-EGFP distribution in HuTu-80 cells revealed numerous
intracellular vesicular-like structures (Figs. 2A and
6A). To measure the dynamics
of these structures and further investigate the role of cytoskeletal
elements in hRFC transport, we employed video-rate confocal imaging to
capture image sequences with sufficient temporal resolution to track
movements of individual vesicles. Experiments were performed at both
37 °C (Fig. 6A) and 22 °C (Fig. 6B), and
results under both conditions are displayed in Fig. 6 as well as in the
supplemental video material (Videos 1 and 2). Fig. 6A shows
a single frame from a video collected at 37 °C (left) and
illustrates the method used to track the motion of hRFC-EGFP-containing
structures (right). The coordinates of discrete fluorescent
structures were tracked at 66-ms intervals (two-frame averages) to
generate color-coded tracks representing movements of several vesicles
throughout the imaging period (Fig. 6A, right).
Fig. 6B shows similar processing applied to a cell maintained at 22 °C from which several tracks have been expanded to
illustrate generalized aspects of hRFC-EGFP dynamics.
Four general features were evident from cells incubated at both
temperatures. First, hRFC-EGFP was localized within structures of
varied size and shape (diameters ~0.3-1.6 µm), including spherical as well as tubular-like vesicles (Videos 1 and 2). Second, there was a
wide variability in the dynamics of hRFC-containing vesicles. Some
showed little motion (e.g. vesicle a, Fig.
6B), whereas others remained highly dynamic over the entire
imaging period or displayed interspersed periods of relatively static
and dynamic behavior (e.g. vesicle b, Fig.
6B). Third, the motion of dynamic vesicles was strikingly
multidirectional rather than showing unidirectional progression toward
the plasma membrane. Vesicles frequently retraced their paths, moved
toward and away from the cell surface (e.g. vesicle
d, Fig. 6B) or tracked circumferentially beneath
the plasma membrane (e.g. vesicle c, Fig.
6B). Finally, vesicles located within the same portion of
the cell often showed significant overlap in their tracks (examples in
Fig. 6, A-C).
The high temporal resolution of these video-rate confocal images
revealed two discrete components to the observed vesicular dynamics,
namely, periods of rapid, approximately linear motion (e.g.
regions marked by black lines for vesicle b, Fig.
6B), interspersed with lengthy periods of relative
immobility during which vesicles displayed Brownian-like movements
constrained within a small area (Fig. 6B). These alternating
patterns of motion were apparent at both temperatures, but at 22 °C
the dwell time of vesicles in the stationary state became longer, and
their velocities during linear motions were slower (Fig.
6C). Further reduction in temperature to 4 °C almost
completely inhibited motility (data not shown).
To quantitate the effect of temperature better, we measured the
velocities of vesicles during periods of rapid, directed linear transport. Measurements at 37 °C from 79 vesicles (15 cells) showed linear movement over a mean distance (run length) of 3.16 ± 0.16 µm at a velocity of 1.56 ± 0.22 µm s
Having determined these parameters under control conditions, we
proceeded to examine the effect of cytoskeletal disruption on vesicle
motion. Video data were recorded before and after addition of the
microtubule disrupting drugs nocodazole (10 µM) or
colchicine (10 µM), with In this study, we employed confocal imaging methods to answer
three questions regarding the cell biology of hRFC. Where is the
full-length hRFC-EGFP fusion protein localized in mammalian epithelial
cells? How does the sequence of the hRFC protein control its cellular
targeting? What cellular mechanism(s) underlie the trafficking of hRFC
to the cell membrane? Our results in each of these areas are discussed
separately below.
Cellular Localization of hRFC-EGFP--
Ligation of the green
fluorescent protein to the COOH terminus of the full-length hRFC
sequence generates a functional fusion protein (11, 21) that we had
shown previously localizes to the plasma membrane of Xenopus
oocytes (11). Here we extended those studies to mammalian cells and
show that hRFC-EGFP similarly targets to the plasma membrane of a human
duodenally derived cell line (HuTu-80; Fig. 2, A and
C) as well as several other epithelial cell lines (Fig.
2E). However, in addition to hRFC-EGFP expressed at the
plasma membrane, intracellular structures also showed appreciable fluorescence (Figs. 2, 3, 6, and 7). The proportion of fluorescent protein in intracellular structures was greatest soon after
transfection and likely represents biosynthetic and trafficking pools
of hRFC-EGFP. Nevertheless, about 30% of cellular hRFC-EGFP was
retained intracellularly (Fig. 6C) even >30 h
post-transfection. The identity of these intracellular structures and
the possibility that they are in exchange with the plasma membrane
compartment are being investigated currently.
Confocal images of the axial distribution of hRFC-EGFP in HuTu-80
monolayers (e.g. Fig. 2C) indicate that hRFC-EGFP
targets to the entire cell surface rather than showing a strongly
asymmetric distribution across the cell. These data suggest that folate
transport can occur over the entire cell surface, consistent with
previous results from in vitro assays (22, 23). However, it
is important to state that the targeting of membrane proteins to
specific cell surface domains is likely dependent on the culture
conditions as well as cell type (24-26), and our results should be
considered only in that context and may not, therefore, reflect the
polarity of hRFC distribution in vivo.
Molecular Determinants of hRFC Targeting in Mammalian
Cells--
We show that the polypeptide domains essential for
targeting hRFC to the plasma membrane reside within the polypeptide
backbone of the protein rather than in the NH2-terminal
(amino acids 1-27) or COOH-terminal (amino acids 452-591) cytoplasmic
portions of the protein. Partial or full truncation of the cytoplasmic
tail of hRFC (hRFC[1-530]-EGFP and hRFC[1-452]-EGFP,
respectively) did not affect the plasma membrane targeting of these
constructs (Fig. 3). Similarly, ablation of the
NH2-terminal sequence (hRFC[28-591]-EGFP) did not
prevent cell surface localization. Further, the doubly truncated
construct (hRFC[19-466]-EGFP), with ablation of both the
NH2-terminal and COOH-terminal tails, still localized to
the plasma membrane (Fig. 3).
Although the NH2- and COOH-terminal cytoplasmic regions are
nonessential for conferring cell surface localization, these regions play accessory roles in modulating the efficiency of expression. For
example, although the NH2-terminal-deleted construct
(hRFC[28-591]-EGFP) reached the cell surface, a considerable portion
of this protein was retained intracellularly (Fig. 3). Further,
measurements of the total fluorescence intensity showed differences
that likely relate to their expression level (Fig. 4). Fluorescence
intensity decreased on full (hRFC[1-452]-EGFP), but not partial
(hRFC[1-530]-EGFP), truncation of the COOH-terminal cytoplasmic tail
(Fig. 4). This consideration could be of clinical significance in that
truncation mutations (14) may affect not only the function and
trafficking of hRFC to the cell surface, but also the rates of
synthesis and degradation of the mutated proteins. Finally, we note
that in the Xenopus oocyte expression system hRFC-EGFP,
hRFC[1-530]-EGFP and hRFC[1-452]-EGFP targeted to the plasma
membrane in a similar manner to that in HuTu-80 cells, with
hRFC[1-452]-EGFP again being expressed at a similarly lower
efficiency (11). Thus, the targeting mechanisms of hRFC appear well
conserved among very different cell types and organisms.
A third important point is that the integrity of the backbone of
hRFC-EGFP is important for export of the polypeptide from the
endoplasmic reticulum to the cell surface. Proteins in which the
COOH-terminal end of the polypeptide backbone was truncated (hRFC[1-301]-EGFP and hRFC[1-144]-EGFP) remained trapped within intracellular membranes (Fig. 3). Similarly, truncation of the NH2-terminal end of the polypeptide backbone
(hRFC[302-452]-EGFP), resulted in low expression and intracellular
localization. Because the constructs hRFC[1-301]-EGFP and
hRFC[302-452]-EGFP, taken together, comprise the entire sequence of
hRFC-EGFP, this suggests that no single contiguous amino acid motif
dictates export to the cell surface. Rather, a more complex interplay
between two or more determinants distributed widely along the
polypeptide backbone appears to be involved.
Sequences critical for plasma membrane targeting have been identified
in many different cell surface proteins. In contrast to our findings
with hRFC, such motifs frequently reside within the cytoplasmic
NH2- or COOH-terminal tails of these proteins (27-32). For
example, truncation of the COOH-terminal tails of the rat liver-rat
Na+/bile acid cotransporter (29), the type iib
NaPi cotransporter (31), or the NH2-terminal
domain of the SGLT1 glucose transporter prevents cell surface
expression of the respective proteins (28). However, these cytoplasmic
regions seem to play a less essential role in directing the cell
surface targeting of hRFC. This observation is not without precedent
within the major facilitator superfamily of transporters (33) because
similar results have been obtained with other proteins that share the
12 membrane-spanning topology of hRFC (21). For example, the
COOH-terminal tail is not essential for cell surface targeting,
stability, or function of the Escherichia coli
H+/lactose permease (34), and total truncation of the
COOH-terminal tail of the glucose transporter GLUT1 impaired function,
but not cell surface localization (35). However, it is probably unwise to generalize about conserved targeting mechanisms among different classes of proteins, especially because of the low sequence similarity between the RFC family and other transporters within this group. Our
data simply underscore the plethora of targeting signals that can act
to route polypeptides to their final cellular destinations.
Intracellular Trafficking of hRFC-EGFP--
Our previous studies
(11) involving expression of hRFC in Xenopus oocytes
indicated that delivery of hRFC-EGFP to the plasma membrane involves
microtubule based transport. Here, we confirm that finding in mammalian
epithelial cells and describe the subcellular dynamics of this
vesicular transport process.
First, the microtubule-disrupting agent nocodazole almost completely
abolished cell surface expression of hRFC-EGFP, whereas disruption of
actin filaments by cytochalasin D was without effect. Second,
measurements of the dynamics of hRFC-containing vesicles yielded
velocities (~1.2-1.5 µm s
A strikingly feature was that movement of hRFC-EGFP-containing vesicles
was multidirectional. Individual vesicles displayed an apparent
haphazard pattern of motion, frequently retracing their steps, despite
the net progression of protein to the cell surface. This
behavior likely results because association with microtubules is only
transient and is interrupted by breaks, with subsequent directional
changes on reassociation with individual microtubules (Fig.
6B). Such multidirectional transport, and especially, rapid
reversal of individual vesicles on single tracks, supports roles for
both plus end- and minus end-directed motors attached to individual
hRFC-EGFP-containing vesicles (36). Further, our observation of
frequent circumferential movements close to the cell surface suggests
that the final stage of insertion, and possibly retrieval, of hRFC-EGFP
from the cell membrane may be a precisely regulated process.
In summary, our results demonstrate that the molecular determinants
responsible for hRFC targeting to the cell surface of mammalian
epithelia are integral to the hydrophobic backbone of the polypeptide,
rather than lying within the NH2- and COOH-terminal cytoplasmic regions. Further, the integrity of the backbone of hRFC is
essential for export of the protein from the endoplasmic reticulum and
its trafficking via microtubules to the cell surface.
We thank Arsalan Hejazi for help with data analysis.
*
This work was supported by the Department of Veterans
Affairs, the University of Minnesota Medical School, and National
Institutes of Health Grants DK-56061 and DK-58057 (to H. M. S.) and
GM-48071 (to I. P.).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.
¶
Both authors contributed equally to this work.
¶¶
To whom correspondence should be addressed.
Tel.: 562-826-5811; Fax: 562-826-5731; E-mail: hmsaid@uci.edu.
Published, JBC Papers in Press, June 26, 2002, DOI 10.1074/jbc.M205955200
The abbreviations used are:
RFC, reduced folate
carrier;
hRFC, human reduced folate carrier;
EGFP, enhanced green
fluorescent protein;
EYFP, enhanced yellow fluorescent protein;
HEK, human embryonic kidney.
Intracellular Trafficking and Membrane Targeting
Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial
Cells*,
§¶,
**,, and**¶¶
Neurobiology and Behavior,
Medicine, and ** Physiology and Biophysics, University
of California, Irvine, California 92697, the § Department of
Pharmacology, University of Minnesota, 55455, and the
Department of Veterans Affairs Medical
Center, Long Beach, California 90822
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lumicolchicine were from Calbiochem.
Tissue culture cell lines were obtained from ATCC (Manassas, VA). All other reagents were obtained from Sigma or from suppliers outlined previously (11, 12).
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Fig. 1.
Construction of hRFC-EGFP fusion
proteins. A schematic representation of the amino acid sequence of
the full-length hRFC-EGFP fusion protein (top) together with
nine truncated fusion constructs (bottom) is shown. Regions
containing the NH2-terminal cytoplasmic region (residues
1-27, dotted line), transmembrane backbone (black
bar), and COOH-terminal cytoplasmic tail (residues 452-591,
striped line) are indicated relative to the sequence of
full-length hRFC. In each construct EGFP was fused to the COOH terminus
of the protein.
Combination of primers used for preparation of the different constructs
by PCR
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Distribution of hRFC-EGFP in
epithelial cells. A, confocal images (x-y)
showing two adjacent HuTu-80 cells expressing hRFC-EGFP, imaged 48 h after transient transfection. B, fluorescence distribution
in a HuTu-80 cell transfected with EGFP alone. C and
D, axial confocal sections (x-z) of the same
HuTu-80 cells shown in A and B, expressing
hRFC-EGFP (C) or EGFP alone (D). E,
distribution of hRFC-EGFP in a variety of different mammalian
epithelial cell lines, each imaged 48 h after transient
transfection.
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Fig. 3.
Cellular distribution of hRFC constructs in
HuTu-80 cells. The montage shows a series of representative
lateral (x-y) confocal sections from HuTu-80 cells
transfected 48 h previously with the indicated constructs. The
laser power was adjusted individually to obtain final images of
equivalent brightness, and these do not therefore reflect differences
in efficiency of protein expression among the constructs. Constructs
are grouped into categories (A-D) as described under
"Experimental Procedures." E, colocalization of EGFP and
FM4-64 fluorescence as a method of quantifying the extent of expression
of different hRFC constructs at the plasma membrane. Images illustrate
HuTu-80 cells transfected with hRFC-EGFP (top) and
hRFC[1-301]-EGFP (bottom). Panels on the
left were obtained using a green band pass filter to show
only EGFP fluorescence. Panels in the middle were
obtained using a red long pass filter to show distribution of the red
fluorescent dye FM4-64 in the plasma membrane. The right
panels show overlays of the red and green
images, with regions of colocalization appearing yellow.
F, bar graphs showing values of colocalization between
FM4-64 and EGFP fluorescence for each of the indicated constructs.
Schematic diagrams on the right illustrate the structures of
each construct (NH2 terminus, white dotted line;
transmembrane domain, yellow bar; COOH terminus, white
line; GFP, green arrow). Data are the mean of values
from 
20 cells.
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Fig. 4.
Efficiency of expression of hRFC-EGFP with
different constructs. Histogram bars show the mean total
fluorescence of HEK-293 cells transfected with different constructs,
expressed as a percentage of that obtained with the full-length
hRFC-EGFP construct and measured on a flow cytometer (mean of at least
three independent experiments).
15 cells) failed to show any further increase
(Fig. 5C).
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Fig. 5.
Effect of cytoskeletal disruption on delivery
of hRFC-EGFP to the plasma membrane. A, distribution of
hRFC-EGFP in HEK-293 cells imaged at different times after transfection
with the full-length construct. B, dual color overlay of
confocal images of hRFC-EGFP-expressing HEK-293 cells obtained 5 h
(left) and 30 h (right) after transfection.
The distribution of hRFC-EGFP is shown in green, and the
plasma membrane was labeled in red by the lipophilic dye
FM4-64. Overlap (colocalization) between these two labels is depicted
in yellow. C, relative distribution of various
EGFP constructs in the plasma membrane, assayed by colocalization of
the green fluorescence with the red membrane marker, as illustrated in
B. Separate culture dishes of HEK-293 cells were transfected
with hRFC-EGFP (black squares), EGFP alone (green
triangles), or hRFC[1-301]-EGFP (red circles), and
colocalization was measured at various times after transfection.
Each point is a mean from 10 cells. D, effect of
cytoskeletal disruption on hRFC-EGFP expression at the cell surface.
Cytochalasin D or nocodazole (both at 400 nM) were added to
cells 12 h after transfection, and the colocalization between
hRFC-EGFP and FM4-64 fluorescence was estimated subsequently after
20 h (dotted blue line in C). Values
represent the relative change in cell surface expression of hRFC-EGFP
over this period (i.e. 1 = no change). Control
measurements (hRFC-EGFP) are shown from culture dishes imaged in
parallel which were not treated with drug.
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Fig. 6.
Vesicular dynamics of hRFC-EGFP resolved by
video-rate confocal imaging. A, left,
average of two consecutive video confocal frames, showing the
distribution of hRFC-EGFP-containing vesicles close to the basal
membrane of a HuTu-80 cell maintained at 37 °C. The video sequence
from which this image was obtained is available as supplementary
material (Video 1). Right, tracks of individual vesicles
(represented by different colors) derived from the video.
B, tracks from a HuTu-80 cell at 22 °C. Individual tracks
(a, b, c, and d, identified
by colors) are shown expanded to illustrate examples of a
relatively static vesicle (a), a vesicle exhibiting rapid
linear movements (lines) interspersed with periods of
immobility (b), a vesicle showing lateral movements beneath
the plasma membrane (c), and a vesicle undergoing multiple
changes in direction highlighted by changes in symbol at successive
reversals (c). C, expanded regions (~6 µm
square) from indicated areas in A and B to
illustrate point-to-point vesicular motion at 37 °C
(left) and 22 °C (right). Points are plotted
every 200 ms (every three data points). D, graphs show mean
velocities during linear motions (left), percentage of time
at rest (middle), and run lengths (right) of
vesicles in cells maintained at 37 °C (red) and 22 °C
(blue). Data are from >50 vesicles in 11 cells.
1 compared
with an average velocity of 0.66 ± 0.07 µm s1
over a linear distance of 2.99 ± 0.42 µm at 22 °C (Fig.
6D). Further, the dwell time at rest was 70.4 ± 3.6%
at 22 °C, decreasing to 49.8 ± 5.1% at 37 °C (125 vesicles, 9 cells, Fig. 6D).
-lumicolchicine (100 µM) as a negative control, and the actin-disrupting agent
cytochalasin D (10 µM). Videos were taken at 5-min
intervals after drug addition, and vesicular dynamics during linear
motions were analyzed as above. Observation of cells for up to 30 min
(30-s imaging periods every 5 min) showed that exposure to the laser
scan had little deleterious effect on vesicular dynamics (average
velocities at the start and end of the 30 min recording were 1.82 ± 0.4 µm s1 versus 1.65 ± 0.4 µm
s1, respectively; 8 cells, Fig.
7A). In contrast, incubation
of cells with 10 µM nocodazole inhibited the linear
motion of vesicles within 5 min (n = 15 cells, Fig. 7,
A and B). Colchicine had a similar effect, albeit
over a slower time course. After a 5-min incubation in 10 µM colchicine, the linear motion of vesicles had
decreased to 0.66 ± 0.07 µm/s (from 11 cells), and by 20 min motion was inhibited (Fig. 7A). -Lumicolchicine, even at
concentrations as high as 100 µM for 30 min, failed to
inhibit vesicular motility (linear velocity of 1.69 ± 0.23 µm/s, Fig. 7A). Similarly, the actin-destabilizing drug
cytochalasin D was without effect on the velocity (1.47 ± 0.22 µm/s; 15 cells) of linear vesicular movement (Fig. 7, A
and C). Representative tracks from cells treated with
nocodazole and cytochalasin D are shown, respectively, in Fig. 7,
B and C, and the videos from which they are
derived are available as supplemental material (Videos 3 and 4).
View larger version (23K):
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Fig. 7.
Effect of cytoskeletal drugs on dynamics of
hRFC-containing vesicles. A, velocities of
hRFC-EGFP-containing vesicles measured at various times after the
addition of 10 µM nocodazole, 10 µM
colchicine, 100 µM -lumicolchicine, and 10 µM cytochalasin D. Bars show mean velocities
(>20 vesicles in 5 cells), measured during rapid, linear movements,
at 37 °C. Asterisks indicate cases in which vesicle
movements were too small to compute velocities. B and
C, representative vesicle tracks in cells treated,
respectively, with 10 µM nocodazole for 5 min and 10 µM cytochalasin D for 5 min. Videos from which these
tracks were derived are available as supplemental material (Videos 3 and 4, respectively).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) and linear run lengths
(average of ~3 µm, ranging up to ~8 µm) consistent with values
reported for microtubule-based vesicular motion in a variety of other
cell types (36, 37) and with the speeds of the microtubule-based motors
kinesin and dynein measured in vitro (38). For example,
microtubule-based transport of GLUT4 vesicles in adipocytes (albeit
resolved at ~10-fold slower frame rates) occurs with linear
velocities of ~0.8 µm s1 over distances of 5-9 µm
(39). Similarly, microtubule-dependent velocities of
exocytotic vesicles in Chinese hamster ovary cells were measured to be
~1 µm s1 with run lengths of ~3 µm (36). Finally,
nocodazole rapidly inhibited the rapid, linear movements of
hRFC-EGFP-containing vesicles (Fig. 7). This effect was mimicked by
colchicine but not -lumicolchicine, a non-tubulin-binding
analog (19). Actin-destabilizing drugs were without effect (Fig.
7).
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at www.jbc.org)
contains Videos 1-4.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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