From the Departments of Neurobiology and Behavior,
Medicine, § Physiology and Biophysics,
University of California, Irvine, California 92697, the
** Department of Pharmacology, University of Minnesota,
Minnesota 55455, and the
Department of
Veterans Affairs Medical Center, Long Beach, California 90822
Received for publication, October 18, 2002, and in revised form, November 13, 2002
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ABSTRACT |
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The human thiamine transporter hTHTR1 is involved
in the cellular accumulation of thiamine (vitamin B1) in many
tissues. Thiamine deficiency disorders, such as thiamine-responsive
megaloblastic anemia (TRMA), which is associated with specific
mutations within hTHTR1, likely impairs the functionality and/or
intracellular targeting of hTHTR1. Unfortunately, nothing is known
about the mechanisms that control the intracellular trafficking or
membrane targeting of hTHTR1. To identify molecular determinants
involved in hTHTR1 targeting, we generated a series of hTHTR1
truncations fused with the green fluorescent protein and imaged the
targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was
functionally expressed at the plasma membrane, analysis of the
truncated mutants demonstrated an essential role for both
NH2-terminal sequence and the integrity of the
backbone polypeptide for cell surface expression. Most notably,
truncation of hTHTR1 within a region where several TRMA truncations are
clustered resulted in intracellular retention of the mutant protein.
Finally, confocal imaging of the dynamics of intracellular hTHTR1
vesicles revealed a critical role for microtubules, but not
microfilaments, in hTHTR1 trafficking. Taken together, these results
correlate hTHTR1 structure with cellular expression profile and reveal
a critical dependence on hTHTR1 backbone integrity and
microtubule-based trafficking processes for functional expression of
hTHTR1.
Thiamine (vitamin B1) is a water-soluble micronutrient that is
essential for many cellular functions relating to growth and development. For example, thiamine pyrophosphate (the coenzyme form) is
important for normal carbohydrate metabolism and energy production (1,
2). Thiamine is not synthesized by humans and other mammals and is
obtained from dietary sources via absorption into intestinal epithelia.
Deficiencies in cellular thiamine accumulation lead to cardiovascular
and neurological disorders and are associated with the inherited
condition of thiamine-responsive megaloblastic anemia
(TRMA)1 (1, 3-7). Recent
molecular analyses have identified the protein product of the
SLC19A2 gene in humans (chromosome 1q23.3) as the site of
mutations that link with TRMA inheritance (2, 7-10). SLC19A2 encodes a saturable, high affinity human thiamine
transporter known as hTHTR1 (2, 7-10), which is expressed in many
tissues, including duodenal epithelium (11). The functional properties of hTHTR1 mimic the biochemical characteristics of thiamine uptake in
intestinal preparations (reviewed in Refs. 12 and 13). The full-length
hTHTR1 protein encodes a 497-amino acid polypeptide with 12 predicted
transmembrane-spanning segments and cytoplasmic NH2- and
COOH-terminal regions (2, 8). Several clinically identified mutations
have been characterized in hTHTR1 (2, 7-10, 14), including ten
prematurely truncated mutants, resulting from either point mutation,
deletion, or insertion of nucleotides (reviewed in Refs. 7 and 15).
Unfortunately, because nothing is known about the molecular
determinants that control the intracellular trafficking and cell
surface targeting of hTHTR1, the precise cellular defect of such
clinically isolated mutants remains undefined.
Through analogy with other nutrient transporters, the intracellular
trafficking and cell surface targeting processes likely involve
specific molecular "motifs" within the amino acid sequence of
hTHTR1. Such determinants can either be localized within specific regions of the polypeptide or result from protein conformation generated from sequences throughout the entire polypeptide sequence (16, 17). To identify regions of hTHTR1 important for cell surface
targeting, we applied confocal imaging techniques to monitor both the
steady-state distribution and real-time trafficking of seven hTHTR1
fusion proteins tagged with the enhanced green fluorescent protein
(EGFP). We have chosen a human duodenal cell line (HuTu-80) for these
studies, because hTHTR1 is expressed at higher levels in human duodenum
than other intestinal epithelia (11). Our results show that (i)
integrity of both the NH2-terminal sequence and the
transmembrane backbone play an important role in the cell surface
expression of hTHTR1; (ii) truncation of the backbone of hTHTR1 between
the sixth and seventh transmembrane domains, at a locus where several
clinical TRMA mutations cluster (7, 15), results in intracellular
retention of hTHTR1; (iii) the cytoplasmic COOH-terminal domain of
hTHTR1 is not required for plasma membrane targeting; (iv) the
intracellular trafficking dynamics of hTHTR1 are critically dependent
on an intact microtubules but not microfilaments. These results
highlight how hTHTR1 is delivered to the cell surface of mammalian
epithelia within a physiologically relevant context and define the
regions of hTHTR1 in which mutation would likely impair thiamine
absorption through disruption of normal cellular targeting mechanisms.
Materials--
[3H]Thiamine (specific activity of
555 GBq/mmol) was purchased from ARC (St. Louis, MO). FM4-64 was from
Molecular Probes (Eugene, OR). The enhanced green fluorescent protein
vector (EGFP-N3) was from BD Biosciences (Palo Alto, CA). Cytochalasin
D, nocodazole, colchicine, and Generation of hTHTR1-EGFP and Truncated Constructs--
cDNA
of the full-length hTHTR1 and truncated constructs were generated by
PCR using the primer combinations shown in Table I and conditions specified previously
(18, 19). Both the PCR products and the EGFP-N3 vector were digested
with the restriction enzymes BamHI and XhoI, and
the products were gel-isolated and then ligated together to generate
in-frame fusion proteins with enhanced green fluorescent protein (EGFP)
fused to the COOH terminus of each construct. The nucleotide sequence
of each construct was confirmed by sequencing. A schematic
representation of each construct is shown below in Fig. 1.
Expression of hTHTR1-EGFP in Xenopus Oocytes--
Capped cRNA
for oocyte microinjection was synthesized from linearized plasmid DNA
using the mMessage mMachine in vitro transcription kit
(Ambion, Austin, TX). Pigmented oocytes (stages V-VI) were isolated
and defolliculated using standard methods (19-21) and then
microinjected with either cRNA into the cytoplasm or cDNA into the
nucleus. Injected oocytes were separated individually into 96-well
plates and maintained in Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.83 mM MgSO4, 0.33 mM
Ca(NO3)2, 0.41 mM
CaCl2, 10 mM HEPES, 550 mg/liter sodium
pyruvate; 0.05 mg/ml gentamicin, pH 7.4, at 18 °C) with repeated
solution changes at least every 18 h. Additional oocytes from the
same donor were injected with water as parallel controls for both
viability and [3H]thiamine uptake assays. For antisense
studies, an equal amount of hTHTR1 cRNA was injected into two batches
of oocytes, one batch of which was co-injected with a
5'-cholesterol-modified DNA oligonucleotide (5'-CATCCATCCGGGGCGCGAGGGGAGG) complementary to a sequence upstream of
the hTHTR1 start codon (-18 to +7 base pairs).
[3H]Thiamine uptake experiments were performed at least
48 h after injection by incubating six to eight oocytes for 1 h at room temperature in Barth's solution (200 µl) supplemented with
150 nM [3H]thiamine. Uptake was
terminated by several washes in ice-cold Barth's solution (5 ml),
after which individual oocytes were transferred to scintillation vials
and dissolved in 10% SDS (250 µl) before scintillation counting.
Cell Culture and Transient Transfection--
Human
duodenal-derived intestinal epithelial cells (HuTu-80), opossum kidney
cells (OK), human embryonic kidney cells (HEK-293), human hepatoma
cells (HepG2), and Madin-Darby canine kidney cells (MDCK) were
maintained in minimal essential medium, supplemented with 10% fetal
bovine serum, glutamine (0.29 g/liter), sodium bicarbonate (2.2 g/liter), penicillin (100,000 units/liter), and streptomycin (10 mg/liter). For transient transfection, cells were grown on sterile
glass-bottomed Petri dishes (MatTek, MA) and transfected at 90%
confluency with 1 µg of plasmid DNA using LipofectAMINE 2000 (Invitrogen, CA). After 24 h, cells were analyzed by confocal microscopy.
Generation of Stable Cell Lines--
Three stable cell lines
were generated by selection of hTHTR1-EGFP, hTHTR1-pcDNA3.1, and
EGFP-N3 expressing HuTu-80 cells with G418 (0.8 mg/ml) in minimal
essential medium supplemented with 10% fetal bovine serum. Molecular
analyses of stable cell lines were performed using semi-quantitative
RT-PCR. Total RNA (5 µg) was isolated from stably transfected and
untransfected HuTu-80 cells and used to generate first-stranded
cDNA with hTHRT1 gene-specific primers (Table I) and a
superscript First-Strand synthesis kit (22). After subsequent PCR
reactions, agarose gels (0.7%) were stained with ethidium bromide, and
the intensity of the product band was quantified by densitometry. For
[3H]thiamine uptake experiments, cell monolayers were
incubated for 3 min at 37 °C in Krebs-Ringer buffer (pH 7.4)
supplemented with [3H]thiamine (30 nM).
Uptake was terminated by addition of ice-cold Krebs-Ringer buffer, and
accumulated radioactivity was measured by scintillation counting.
Confocal Microscopy--
Cells were imaged using either a
customized laser scan confocal microscope (23) or a video-rate scanning
confocal microscope (24). Both microscopes were based on an Olympus
IX70 inverted microscope fitted with a 40× oil-immersion objective.
Fluorophores were excited using the 488-nm line from an argon
ion-laser, and emitted fluorescence was monitored with a 530 ± 20-nm band pass (EGFP) or a 650-nm long-pass filter (FM4-64). Using
the linescan microscope (Figs. 2A, 3, 4, and 5A),
frame scans were obtained by scanning the laser line either laterally
(x-y scan) or axially (x-z scan) within the cell.
For real-time images of vesicular trafficking, hTHTR1-EGFP fluorescence
was monitored using the video-rate confocal microscope (Figs. 6 and 7).
Petri dishes were maintained at 22 °C or 37 °C using a
thermostatted stage mount. Images were captured at 30 Hz over a 40-µm
square region using a video-acquisition board/frame grabber and
archived onto digital tape. For image processing, video data were
digitized into image stacks using the stream acquisition function of
the Metamorph imaging package (Universal Imaging, Downingtown, PA). Raw
data stacks were averaged (as merges of two sequential 33-ms frames) and low pass-filtered before analysis. Frame-to-frame particle tracking
was performed using customized macro routines in Metamorph to generate
two-dimension (x-y) tracks of discrete particles over time.
From such plots, quantitative data concerning the linear velocities and
associated vectorality of individual structures could be analyzed
throughout the entire recording period. Quick-time movies of video
sequences are appended as supplementary material (videos 1-5).
Design of hTHTR1 Truncations--
The schematic representation of
the full-length hTHTR1-EGFP fusion protein in Fig.
1A illustrates the structural
organization of the protein, comprising an NH2-terminal
cytoplasmic region (amino acid residues 1-29), a transmembrane domain
with 12 predicted transmembrane-spanning regions (amino acid residues
30-479), and a COOH-terminal cytoplasmic domain (amino acid residues
480-497) that was fused to EGFP. To identify regions important for
cell surface expression, we generated a series of seven truncated
fusion proteins (Fig. 1, B and C). First, we
analyzed the role of NH2-terminal, transmembrane backbone,
and COOH-terminal regions in hTHTR1 targeting (Fig. 1B).
Second, we examined the effect of premature truncation between the
sixth and seventh transmembrane domains of hTHTR1 (Fig. 1C),
where three premature truncations (amino acid residues 239, 250, and
259) occur in clinical presentations of TRMA (7, 15).
Functionality of the hTHTR1-EGFP--
We first confirmed the
functionality and appropriate cellular targeting of the full-length
hTHTR1-EGFP fusion construct by expressing the protein in
Xenopus oocytes following nuclear injection of cDNA
encoding hTHTR1-EGFP (Fig. 1A). Two days after
microinjection, oocytes were imaged by confocal microscopy for
hTHTR1-EGFP expression. In all oocytes examined (n = 8 cells), hTHTR1-EGFP fluorescence was evident with an average 11.0 ± 2.5-fold greater peak fluorescence in the animal compared with the
vegetal pole (Fig. 2A).
Fluorescence was confined to a radial band 5.2 ± 1.4 µm wide,
consistent with the thickness of the invaginated oocyte plasma membrane
(19). To confirm localization of hTHTR1-EGFP to the cell surface, and to compare the functionality of hTHTR1-EGFP relative to hTHTR1, we
measured [3H]thiamine uptake in Xenopus
oocytes heterologously expressing either construct. Oocytes expressing
hTHTR1 or hTHTR1-EGFP exhibited [3H]thiamine accumulation
(108.0 ± 9.6 and 131.5 ± 35.5 fmol/oocyte/h, respectively)
that was about double that in water-injected controls (53.0 ± 1.0 fmol/oocyte/h) or oocytes co-injected with hTHTR1 cRNA and an antisense
oligonucleotide to prevent translation of hTHTR1 (58.6 ± 2.4 fmol/oocyte/h). These values for [3H]thiamine uptake in
cells expressing hTHTR1-EGFP (~1.5-fold uptake) were similar to those
reported elsewhere on expression of hTHTR1 cDNA (2, 14). Thus, the
functionality of the hTHTR1-EGFP fusion protein was not impaired by
COOH-terminal fusion to GFP, and the fusion protein correctly targeted
to the cell surface.
Plasma Membrane Localization of hTHTR1-EGFP in Epithelial Cell
Lines--
To study the targeting of hTHTR1-EGFP in mammalian
epithelia, cDNA encoding hTHTR1-EGFP was transiently transfected
into a variety of epithelial cell lines, and the resulting fluorescence distribution was analyzed by confocal microscopy. In HuTu-80 cells, hTHTR1-EGFP expression was evident in the cell membrane as well as in
processes extending from the cell surface and a variety of
intracellular structures (Fig.
3A). This distribution
differed markedly from HuTu-80 cells transfected with EGFP alone, in
which the entire cytoplasmic volume was fluorescence (Fig.
3B). These contrasting distributions were also apparent in
axial scans (side views) of HuTu-80 cells expressing either
hTHTR1-EGFP or EGFP alone (Fig. 3, C and D,
respectively). A similar plasma membrane targeting of hTHTR1-EGFP was
also apparent in several other cell lines, including MDCK, OK, HEK-293,
and HepG2 (Fig. 3E).
Determination of Regions of hTHTR1 Important for Cell Surface
Targeting--
To investigate the role of specific regions of hTHTR1
structure in cell surface targeting, we constructed a series of hTHTR1 truncations (Fig. 1, B and C) and compared their
expression profile with the full-length hTHTR1-EGFP fusion protein.
Fig. 4 (A-C) shows
representative confocal images (x-y scans) of HuTu-80 cells imaged 48 h after transient transfection with each construct. Several expression patterns were apparent, which could be grouped into
three classes: 1) expression predominantly at the cell surface (hTHTR1-(1-479)-EGFP and hTHTR1-(19-486), Fig. 4A); 2) a
cytosolic, non-membrane-bound expression (hTHTR1-(1-29)-EGFP and
hTHTR1-(480-497)-EGFP, Fig. 4B), and 3) expression
confined within intracellular membranes (hTHTR1-(1-240)-EGFP,
hTHTR1-(241-479)-EGFP, and hTHTR-(30-497)-EGFP, Fig.
4C).
Targeting of different hTHTR1-EGFP constructs to the cell surface was
quantified utilizing a distribution overlap assay (18, 25), in which
cells transfected with individual hTHTR1-EGFP fusion constructs were
co-stained by extracellular application of the red
lipophilic marker FM4-64 to selectively stain the plasma membrane.
Fig. 4 illustrates this method for cells expressing hTHTR1-EGFP (Fig.
4D) and hTHTR1-(1-240)-EGFP (Fig. 4E). The green fluorescence of hTHTR1-EGFP overlapped considerably with the
red fluorescence of FM4-64 to yield a yellow
color indicating a high degree of localization of hTHTR1-EGFP at
the cell surface (Fig. 4D). In contrast, little fluorescence
co-localization was observed with hTHTR1-(1-240)-EGFP (Fig.
4E), which remained localized within intracellular
membranes. Fig. 4F shows collated measurements of fluorescence co-localization obtained by this method for hTHTR1-EGFP and all seven truncated constructs. A high degree (~60%) of
fluorescence overlap was observed in cells expressing hTHTR1-EGFP,
hTHTR1-(1-479)-EGFP, and hTHTR1-(19-486)-EGFP, confirming that each
of these constructs targeted to the cell surface. Little
co-localization (~10%) was seen with the remaining constructs
confirming their inability to reach the plasma membrane.
Establishment of a Stable HuTu-80 Cell Line for Analysis of
hTHTR1-EGFP Trafficking--
To facilitate investigation of the role
of the cytoskeleton in controlling the delivery to hTHTR1-EGFP to the
cell surface, we generated a stably expressing HuTu-80 cell line. Fig.
5A shows a confocal image of a
HuTu-80 monolayer after 8 weeks of antibiotic selection, when the
majority of cells (~60%) exhibited the characteristic cell surface
and intracellular distribution of hTHTR1-EGFP. Two further stable cell
lines, expressing either hTHTR1 or EGFP alone, were established in
parallel as controls. RT-PCR analysis of all three stable cell lines
showed that hTHTR1 mRNA expression was ~5-fold greater in both
the hTHTR1 and hTHTR1-EGFP stable HuTu-80 clones compared with the
EGFP-expressing stable cell line or mock transfected HuTu-80 cells
(Fig. 5B). Furthermore, uptake of [3H]thiamine
in both the hTHTR1-EGFP (113 ± 6 fmol/mg of protein/3 min) and
hTHTR1 (128 ± 3 fmol/mg of protein/3 min) stable cell lines was
significantly greater than in the EGFP stable cell line (73 ± 5 fmol/mg of protein/3 min) or in untransfected HuTu-80 cells (59 ± 4 fmol/mg of protein/3 min). Taken together, these data further support
the functionality of the hTHTR1-EGFP fusion construct and establish the
validity of this stable cell line for analyses of hTHTR1-EGFP
properties.
Intracellular Trafficking Dynamics of hTHTR1--
Confocal imaging
of the stable hTHTR1 HuTu-80 cell line revealed numerous vesicular-like
structures within the cytoplasm (Figs. 5A and
6A). The dimensions of these structures ranged between 0.5 and 1.5 µm (Fig. 6B),
suggesting that hTHTR1-EGFP localized within a heterogeneous population
of intracellular vesicles. Vesicles were evident throughout the
cytoplasm, but fluorescence was most concentrated close to the nucleus,
which presumably represents the initial sites of hTHTR1-EGFP
biosynthesis (see "Discussion"). To investigate the dynamics of
individual vesicles trafficking toward the cell surface, we employed
video-rate confocal imaging to capture image sequences with sufficient
temporal resolution (30 frames per second) to track the movement of
single vesicles in real time. An analysis of the video data
(supplementary video 1), illustrated by a single image frame in Fig.
6A, is shown schematically in Fig. 6C. The
coordinates of fluorescent vesicles were tracked at 66-ms intervals
(two-frame averages) to generate color-coded tracks depicting their
motion. Several tracks are shown expanded in Fig. 6C and
illustrate three generalized features of hTHTR1-EGFP dynamics.
First, in any given cell, the population of hTHTR1-EGFP-containing
vesicles exhibited a wide variation in motility, some vesicles remained
predominantly static (e.g. vesicle iv, Fig.
6C), whereas others were highly dynamic (e.g.
vesicles i, ii, and iii, Fig. 6C). The distribution between the "dynamic" and
"static" phenotype was temperature-dependent, with
progressively more vesicles becoming motionless at lower temperature
(data not shown). Furthermore, the velocity of vesicular movements
increased with temperature. At 22 °C, the average vesicular velocity
(including stationary periods) was 0.43 ± 0.13 µm
s
Second, the high temporal resolution of the video-rate confocal records
revealed two discrete components to the dynamics of individual
vesicles, namely periods of rapid, approximately linear motion
(e.g. black lines, vesicle iii, Fig.
6C) interspersed with periods of relatively immobility
during which vesicles displayed small Brownian-like movements
(e.g. arrow, vesicle iii, Fig. 6C). Because of this "stop-start" behavior, "average velocity"
measurements presented above underestimate the peak velocity of
hTHTR1-EGFP-containing vesicles. Therefore, we quantified velocities
during the periods of rapid, directed linear movements (Fig.
6D). At 37 °C the average linear velocity was 1.71 ± 0.13 µm/s, decreasing to 0.73 ± 0.36 µm/s at 22 °C
(n = 38 vesicles, 5 cells). However, at both
temperatures the "run length" (the length of linear segments) was
similar, 2.75 ± 0.22 µm at 22 °C and 3.22 ± 0.22 µm
at 37 °C.
Third, the movement of vesicles was strikingly multidirectional. Rather
than displaying a consistent progression toward the cell surface from
the interior of the cell, vesicles frequently retracted their steps
(e.g. vesicle i, Fig. 6C). Similar
behavior was also apparent when vesicles were in close proximity to the cell boundary, where they appeared to track cirumferentially beneath the plasma membrane (e.g. vesicle ii, Fig.
6C). Despite the multidirectional nature of vesicular
motion, vesicles moving through the same area of the cytoplasm showed
significant overlap in their tracks (Fig. 6C).
Role of Microtubules and Actin Microfilaments in hTHTR1-EGFP
Trafficking--
Having quantified the characteristics of vesicular
motion at 37 °C (Fig. 6D), we proceeded to examine the
effect of cytoskeletal disruption on the motion of individual
hTHTR1-EGFP-containing vesicles. Image sequences were recorded before
and after addition of either the microtubule disrupting drugs
nocodazole or colchicine (with
Fig. 7 shows the effect of incubation of
HuTu-80 cells with nocodazole (Fig. 7A, 10 µM
for 5 min), colchicine (Fig. 7B, 10 µM for 15 min), In this study, we investigated the physiological behavior of
hTHTR1 within intestinal epithelial cell lines with regard to three
issues. First, what regions of the hTHTR1 polypeptide are important for
targeting hTHTR1 to the cell surface? Second, how do clinically
relevant hTHTR1 truncations affect the cellular targeting of hTHTR1?
Third, what cytoskeletal elements mediate hTHTR1 trafficking? To
resolve these questions, we employed confocal microscopy to image the
dynamics of hTHTR1-EGFP fusion proteins. This strategy has proved a
powerful approach to monitor the targeting and expression of nutrient
transporters and other proteins, because it allows localization with
high spatial and temporal resolution in live cells (18, 25, 26).
Heterologous expression of hTHTR1-EGFP in either mammalian epithelial
cell lines (Figs. 3 and 5A) or the Xenopus oocyte
expression system (Fig. 2A), resulted predominantly in
expression of hTHTR1-EGFP fluorescence at the cell surface, as expected
for a transporter protein, as well as in cellular processes extending
from the plasma membrane and a variety of intracellular structures (see
below). Ligation of EGFP to the COOH terminus of hTHTR1 did not impair
functionality, as assessed by [3H]thiamine accumulation
in both Xenopus oocytes (Fig. 2B), and a stably
expressing human duodenal cell line (Fig. 5C).
Regions of hTHTR1 Important for Cellular Targeting--
The
delivery of proteins to specific cellular compartments depends on the
presence of targeting commands embedded as primary sequence, structural
conformations, or post-translational processing signals within the
protein structure (27, 28). Many "motifs" responsible for directing
nutrient transporters to the plasma membrane of epithelial cells have
been localized within the NH2 terminus (29), COOH terminus
(30), and/or the transmembrane backbone regions of individual proteins
(18). Here we show that both NH2-terminal
sequence (within amino acids 19-29) as well as determinants within the
transmembrane backbone are important in directing the expression and
consequent export of hTHTR1 from the endoplasmic reticulum to the
plasma membrane. This conclusion is based on the following evidence.
First, the COOH-terminal cytoplasmic region (amino acids 480-497) was
unimportant for plasma membrane targeting, because removal of this
region failed to prevent cell surface localization
(hTHTR1-(1-479)-EGFP). In contrast, removal of the
NH2-terminal sequence (amino acids 1-29) prevented cell surface expression of the truncated protein (hTHTR1-(30-497)-EGFP), which was retained within intracellular membranes (Fig. 4C).
Second, the NH2-terminal sequence alone was not sufficient
for cell surface expression, because hTHTR1-(1-240)-EGFP, a construct
in which the entire NH2-terminal sequence was preserved,
but the polypeptide backbone was truncated after six transmembrane
helices, was retained within intracellular membranes. The role of the
polypeptide backbone was underscored by the observation that
hTHTR1-(19-486) in which both the NH2- and COOH-terminal
regions were partially truncated, but the integrity of the
transmembrane domains preserved, was targeted to the cell surface (Fig.
4, A and F). Further experiments are needed to
delimit whether polypeptide backbone integrity per se, or
specific determinants downstream of the truncation site, are needed for
plasma membrane expression. The important point is that, if COOH
determinants exist, they do not function in isolation, because
hTHTR1-(241-479) was also defective at export from intracellular membranes. Most likely, plasma membrane targeting is dependent on both
NH2-terminal sequence and determinants distributed
throughout the hTHTR1 polypeptide backbone.
A final important point relates to our knowledge of targeting
determinants in other members of the SLC19A gene family. As the protein product of the SLC19A2 gene, hTHTR1 shares
significant sequence identity (~40% (2, 8)) with the human reduced
folate carrier (hRFC), the protein product of the SLC19A1
gene (31). Topological analysis of both proteins suggests a similar 12 transmembrane-spanning topology (Refs. 2, 32, and 33, but see Ref. 10).
We recently analyzed the effect of specific domain ablations on hRFC
targeting (18, 19) and demonstrated that polypeptide backbone integrity plays a crucial role in the plasma membrane expression of hRFC. This
result mimics that observed here with hTHTR1, in that the polypeptide
backbone of either transporter (hTHTR1-(19-486)-EGFP or
hRFC-(19-466)-EGFP) with minimal flanking sequence is sufficient to
direct cell surface expression. One subtle difference with hTHTR-1 is
that the NH2-terminal sequence (amino acids 19-29) is more
critical for cell surface expression than the corresponding region in
hRFC (18). This may relate to the difference in net positive charge of
the NH2-terminal domain of hTHTR1 ("+4") compared with
hRFC ("0"), which may be crucial in directing appropriate protein
translocation and folding within the endoplasmic reticulum membranes
(34). Overall these results suggest the possible
conservation of targeting mechanisms between these closely related
cousins within the major facilitator superfamily of transporters (35). It remains to be seen whether this homology extends to hTHTR2, the
protein product of the recently characterized SLC19A3 gene (36, 37), which encodes a second human thiamine transporter with even
greater amino acid residue identity to hTHTR1 (~48% (36, 37)).
Clinical Relevance of Truncations within the hTHTR1 Polypeptide
Backbone--
TRMA is a rare autosomal recessive disorder, attributed
to mutational impairment of hTHTR1 function caused by specific
mutations in the SLC19A2 gene (2, 7-10, 14, 15). At least
fourteen distinct mutations have been identified from TRMA patients (7, 15) of which 10 result in premature truncation of hTHTR1 (Fig. 1A). Three of these truncations (239, 250, and 259 amino
acids (8)) result in premature termination within a region normally between the sixth and seventh transmembrane helices (10). Confocal imaging of hTHTR1-(1-240)-EGFP showed that the truncated protein is
retained within intracellular membranes (Fig. 4C), with no visible cell surface expression (Fig. 4E). Therefore, the
three clinically relevant truncations occurring within this same region likely result in abrogation of cell surface expression. Because the
four upstream truncations (after amino acids 66, 97, 127, and
162) lack further amino acids (Refs. 7, 8, 10, and 15), each of these
truncations likely results in intracellular retention of the protein
product. Further experiments are needed to identify the cellular
localization of the three remaining clinically identified truncations
(313, 358, and 385 amino acids in length) (Refs. 9, 15, and 38),
although we note that similar sized truncations of hRFC backbone
resulted in intracellular retention (18).
Intracellular Trafficking of hTHTR1-EGFP--
Generation of a
stable hTHTR1-EGFP-expressing duodenal cell line facilitated analysis
of hTHTR1-EGFP trafficking as synthesis of hTHTR1-EGFP was ongoing and
the population of intracellular fluorescent structures more numerous
than with transient transfection. The steady-state distribution of
hTHTR1-EGFP was defined by a strong cell surface expression, together
with considerable fluorescence within a juxtanuclear pool, most likely
the Golgi apparatus. Vesicular motility was critically dependent on an
intact microtubular cytoskeleton, as shown by the effects of
pharmacological disruption with nocodazole or colchicine, but not
Summary--
These studies show that, unlike many nutrient
transporters, the molecular determinants that dictate hTHTR1 targeting
to the cell surface are not located within the COOH-terminal
cytoplasmic tail of the polypeptide but are dependent on residues
within both the NH2-terminal region and the polypeptide
backbone. Premature truncation of the polypeptide backbone, as seen in
several independent clinical mutations in hTHTR1, causes intracellular
retention of the protein. Finally, trafficking of hTHTR1 to the cell
surface is critically dependent on intact microtubules but not microfilaments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lumicolchicine, were from
Calbiochem (La Jolla, CA). Antisense oligonucleotides were from Heligen
Laboratories (Huntington Beach, CA). G418 was from Life Technologies
(Invitrogen, CA). Tissue culture cell lines were obtained from ATCC
(Manassas, VA). All other reagents were from Sigma (St. Louis, MO).
Combination of primers used to prepare the different truncated
constructs by PCR
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of hTHTR1 and construction of
fusion proteins. Schematic representation of hTHTR1-EGFP and seven
truncation constructs. In all cases, EGFP was fused to the COOH
terminus of the protein. A, domain organization of amino
acid sequence of hTHTR1-EGFP showing the NH2-terminal
regions (amino acids 1-29); the transmembrane "backbone" (amino
acids 30-479) containing 12 transmembrane spanning regions
(black bars) and the COOH-terminal region (amino acids
480-497). EGFP is illustrated schematically. The positions of ten
clinically identified truncation mutants are shown (open
squares), highlighting the positions of four point mutations
(diagonal crosses) that result in stop codons, and the
lengths of six other prematurely truncated hTHTR1 proteins that result
from upstream nucleotide deletion/insertion events. All ten truncation
mutants have been identified from clinical presentations of TRMA (see
Refs. 7 and 15). B, series of five truncation mutants
designed to investigate the role of NH2- and COOH-terminal
sequence in hTHTR1 targeting. C, two fusion proteins
designed to investigate the role of hTHTR1 backbone integrity in
cellular targeting.
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Fig. 2.
Expression of hTHTR1-EGFP in
Xenopus oocytes. A, bright field image
of a pigmented Xenopus oocyte (far left), to
indicate the orientation of the oocyte in the confocal fluorescence
images. Right, axial (x-z) scans into the animal
(top) and vegetal pole (bottom) hemispheres of a
Xenopus oocyte microinjected 48 h previously with
hTHTR1-EGFP cDNA. Images are scaled to the peak fluorescence
intensity in the animal hemisphere. Oocytes were injected with
hTHTR1-EGFP cDNA to ensure correct processing of the construct
through the endogenous oocyte transcription and translation machinery.
Traces (far right) represent fluorescence
intensity as a function of depth into the oocyte averaged across a
40-µm section of the laser scan line. B, uptake of
[3H]thiamine in oocytes injected >48 h previously with
hTHTR1 cRNA, hTHTR1-EGFP cDNA, water, or hTHTR1 cRNA and an
antisense oligonucleotide. Results represent the mean ± S.E. from
three different donor animals.
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Fig. 3.
Distribution of hTHTR1-EGFP in epithelial
cells. A and B, lateral
x-y confocal images of HuTu-80 cells transiently transfected
with hTHTR1-EGFP (A) or pEGFP alone (B).
C and D, axial confocal sections (x-z)
of HuTu-80 cells expressing hTHTR1-EGFP (C) or EGFP alone
(D). E, lateral confocal images showing
expression of hTHTR1-EGFP in the cell membrane of MDCK (canine kidney),
OK (opossum kidney), HepG2 (human hepatoma), and HEK-293 (human
embryonic kidney) cell lines.
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Fig. 4.
Cellular distribution of truncated
hTHTR1-EGFP constructs in HuTu-80 cells. HuTu-80 cells were
transiently transfected with cDNA encoding the indicated mutants
and lateral (x-y) confocal images were obtained 48 h
later. The laser power was individually adjusted to obtain final images
of equivalent brightness, and differences in intensity do not,
therefore, reflect differences in protein expression between particular
constructs. Constructs are grouped into categories (A-C) as
detailed in the results. D and E, images of
HuTu-80 cells stained with FM4-64 that were previously transfected
with hTHTR1-EGFP (D) and hTHTR1-(1-240)-EGFP (E)
to show green fluorescence of EGFP alone (left),
red fluorescence of FM4-64 in the plasma membrane
(middle), and a dual channel overlay (right).
F, bar graph showing percentage co-localization of EGFP with
FM4-64 fluorescence for each indicated hTHTR1 construct. Data are from
n > 10 transfected cells.
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Fig. 5.
Properties of a HuTu-80 cell line stably
transfected with hTHTR1-EGFP. A, confocal linescan
image (x-y) of hTHTR1-EGFP distribution in a HuTu-80 cell
line maintained for 8 weeks under G418 antibiotic selection.
B, RT-PCR detection of hTHTR1 mRNA from untransfected
(control), as well as three different HuTu-80 stable cell lines.
C, [3H]thiamine uptake assays in mock
transfected and stably transfected HuTu-80 cell lines. Results
represent the mean ± S.E. from n = 3 experiments.
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Fig. 6.
Vesicular trafficking of hTHTR1-EGFP in a
stable HuTu-80 cell line. A, a single frame from a
video-rate confocal image showing the distribution of
hTHTR1-EGFP-containing vesicles in a stably transfected HuTu-80 cell at
37 °C. The entire video sequence is appended as supplementary video
material (video 1). B, dimensions of hTHTR1-EGFP-containing
vesicular structures in the cytoplasm of a stably transfected HuTu-80
cell. Fluorescence profiles of four vesicles are measured along 3-pixel
wide lines at the red and black arrows.
C, tracks of individual vesicle movements schematically
represented from the cell shown in A. For clarity, only a
fraction of the total vesicular tracks are displayed. The high density
of vesicles near the center of the cell (indicated by gray
shading) made it impossible to identify discrete structures within
this region. Individual tracks (i through iv)
have been expanded to provide examples of (i) a vesicle
exhibiting bi-directional movements, (ii) a vesicle tracking
beneath the cell surface, (iii) a vesicle showing rapid
linear movements interspersed by periods of relative immobility, and
(iv) a static vesicle. D, histograms showing
distribution of vesicular velocities quantified at 22 °C
(blue) and 37 °C (red) during periods of
linear movements.
1 (n = 25 vesicles), compared with
0.92 ± 0.21 µm s
1 at 37 °C (n = 93 vesicles).
-lumicolchicine as a negative
control) or the microfilament disrupting drug cytochalasin D.
-lumicolchicine (Fig. 7C, 50 µM for
30 min), or cytochalasin D (Fig. 7D, 10 µM for
30 min), together with the measurements of the effects of each compound
on the linear velocities of hTHTR1-EGFP motion at various times after
drug addition (Fig. 7E). Each diagram of vesicular motion
(Fig. 7, A-D) is associated with a supplementary video
(videos 2-5 in supplemental material) from which the tracking data
were derived. Incubation of cells with nocodazole rapidly inhibited the
linear motion of hTHTR1-EGFP-containing vesicles within 5 min (Fig. 7,
A and E). Colchicine exhibited a similar effect
albeit over a slower time course, with maximal inhibition taking up to
15 min (Fig. 7, B and E). In contrast, addition
of
-lumicolchicine (50 µM) failed to impede vesicular motion, even with incubation periods as long as 30 min (Fig. 7, C and E). Similarly, cytochalasin D had little
effect on the linear motions of hTHTR1-containing vesicles (Fig. 7,
D and E).
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Fig. 7.
Effect of cytoskeletal disruption on the
movement of hTHTR1-EGFP-containing vesicles. Schematic
representations of vesicular dynamics in HuTu-80 cells stably
transfected with hTHTR1-EGFP treated with: A, nocodazole (10 µM, 5 min); B, colchicine (10 µM, 15 min); C, cytochalasin D (10 µM, 30 min); D, -lumicolchicine (50 µM, 30 min). Each image is associated with a video file
attached as supplementary material (videos 2-5). E,
histogram showing the effect of pharmacological treatments on the
observed linear velocities of hTHTR1-EGFP-containing vesicles.
Durations of incubation with each drug concentration are shown
numerically on the x-axis. Asterisks indicate an
inability to measure linear velocities owing to no observed motion.
Data are from n
5 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lumicolchicine as a negative control (Fig. 7). Furthermore,
video-rate confocal measurements of vesicular dynamics (Fig. 6, average
moving velocities of ~1.7 µm/s and run lengths of ~3 µm) are
consistent with microtubule-based transport of different cargos in
other cell types (39, 40), as well as speeds of microtubule-based
motors measured in vitro (41). A striking result was the
observation of multidirectional vesicular motion, both toward and away
from the cell surface, as well as circumferential movements beneath the
plasma membrane (Fig. 6). These images suggest that the trafficking and
insertion of hTHTR1 into the plasma membrane may be physiologically
regulated events, and we are currently investigating mechanisms that
may influence the net progression of hTHTR1 transporters
toward, and removal from, the cell surface.
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ACKNOWLEDGEMENT |
---|
We thank Arsalan Hejazi for help with data analysis.
![]() |
FOOTNOTES |
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* This work was supported by the Department of Veterans Affairs, the University of Minnesota Medical School, and Grants DK-56061 and DK-58057 (to H. M. S.), Grant GM-48071 (to I. P.) and National Service Research Award (NRSA) Fellowship Grant F32DK063750-01 (to V. S. S.) from the National Institutes of Health.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.
The on-line version of this article (available at
http://www.jbc.org) contains videos 1-5.
¶ 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, November 25, 2002, DOI 10.1074/jbc.M210717200
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ABBREVIATIONS |
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The abbreviations used are: TRMA, thiamine-responsive megaloblastic anemia; hTHTR1, human thiamine transporter-1; EGFP, enhanced green fluorescent protein; hRFC, human reduced folate carrier; MDCK, Madin-Darby canine kidney cells; RT, reverse transcriptase.
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REFERENCES |
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