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INTRODUCTION |
Glucose transporters represent a family of proteins that are
responsible for the cellular uptake of glucose and are ubiquitously expressed in mammalian cells in a tissue-specific fashion (1). Normally, these proteins function at the cell surface and recycle between the plasma membrane and intracellular compartment(s) at a rate
that depends on the type of cell as well as the isoform of the
transporter protein. The distribution of glucose transporters between
the plasma and intracellular membranes is strictly regulated, because
this distribution has a major impact on glucose uptake and, hence,
cellular energy metabolism as well as on maintaining glucose
homeostasis in the blood. This is certainly true for the fat and
muscle-specific transporter isoform, Glut4, and, to some extent, for
the "ubiquitous" transporter isoform, Glut1, both of which traffic
in competent cells under tight insulin control (reviewed in Refs. 1 and
2). Glut3, which is expressed to some degree in cultured L6 muscle
cells, has also been shown to redistribute to the plasma membrane after
insulin or insulin growth factor-1 treatment (3). In addition, Glut3 is
expressed in platelets in which it is translocated from
-granules to
the cell surface in response to thrombin, a phenomenon that may reflect increased energy requirements of platelets upon activation (4, 5). The
major Glut3-expressing tissue, however, is brain (1, 6), and it is
unknown whether Glut3 function in brain may be regulated by compartmentalization.
We, therefore, studied the intracellular localization of Glut3 in rat
brain as well as in cultured rat pheochromocytoma PC12 cells. Although
the major part of the Glut3 moiety in both brain and PC12 cells is
localized in the plasma membrane, a fraction of the transporter has
been found inside the cell. Intracellular Glut3 is present in a
biochemically homogenous population of membrane vesicles, which are
co-purified with classical synaptic vesicles but can be separated from
the latter in equilibrium density and velocity sucrose gradients. A
unique feature of Glut3-containing vesicles that allows one to easily
discriminate them from classical synaptic vesicles, which otherwise
have a very similar protein composition (7), is the presence of
aminopeptidase activity. We have determined that this activity
belongs to aminopeptidase B, which has recently been shown to represent
a marker of the regulated secretory pathway in PC12 cells (8). Thus, we
suggest that Glut3-containing vesicles represent a novel secretory
compartment in neurons and neuroendocrine cells. We have also found
Glut3 in clathrin-coated vesicles, suggesting that Glut3 may recycle between the plasma membrane and intracellular membranes via
a clathrin-mediated pathway.
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MATERIALS AND METHODS |
Antibodies--
Affinity purified anti-Glut3 antibody was from
Charles River Pharmservices. Antibody to clathrin heavy chain was from
ICN. Antibody to synaptophysin was from Roche Molecular Biochemicals. Antibodies to SV2 and synaptotagmin were kind gifts from Dr. K. Buckley
(Harvard Medical School). Antibody to synaptobrevin was a kind gift
from Dr. R. Jahn (Max Plank Institute, Gottingen).
Cell Culture--
The pheochromocytoma cell line PC12 was grown
in Dulbecco's modified Eagle's medium supplemented with 5% fetal
bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin in
5% CO2 at 37 °C.
Isolation and Fractionation of Synaptic Vesicles from PC12
Cells--
Confluent 15-cm plates were rinsed twice at 4 °C with
buffer A (150 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM EGTA, 0.1 mM MgCl2). Cells were
scraped from dishes into 1-2 ml of buffer per plate, and homogenized
with 10 strokes in a Dounce homogenizer followed by passage through a
25 gauge needle 10 times. The homogenate was centrifuged at 100 × g for 5 min, followed by centrifugation of the resulting
supernatant (S1) at 27,000 × g for 35 min to yield a
pellet and supernatant (S2) (9). Total intracellular membranes were
pelleted by centrifugation of supernatant S2 at 180,000 × g for 2 h and centrifuged on a 10-30% (w/w) sucrose velocity gradient for 50 min at 48,000 rpm in a SW50.1 rotor. Fractions
were collected and analyzed for synaptic vesicle proteins. Fractions in
the gradient that were enriched in SV2, synaptophysin and
synaptobrevin, were pooled, pelleted, and reapplied on a 10-50% (w/v)
sucrose equilibrium density gradient and centrifuged for 18 h at
48,000 rpm in a SW50.1 rotor. Fractions were collected starting from
the bottom of the gradient and blotted for GLUT3 as well as for
individual synaptic vesicle proteins.
Isolation and Fractionation of Synaptic Vesicles from Rat
Brain--
Synaptosomes were prepared from rat brains according to the
procedure of Huttner et al. (10). The resulting synaptosomal pellet was diluted with 9 volumes of ice cold H2O
(hypotonic lysis of synaptosomes to release synaptic vesicles) and
immediately homogenized with three strokes in a Dounce homogenizer. 1 M HEPES, pH 7.4, was added to a final concentration of 7.5 mM HEPES, and the homogenate was centrifuged for 20 min at
25,500 × g to remove the synaptosomal membrane. The
supernatant was centrifuged for 2 h at 48,000 rpm, in a Ti60
rotor, and the resulting pellet was resuspended in 1 ml of 30 mM sucrose, 4 mM HEPES, pH 7.4, and homogenized
by passage through a 25 gauge needle 5 times back and forth. This
material was loaded on a continuous gradient of 50-800 mM
sucrose, 4 mM HEPES, pH 7.4, and the gradient was
centrifuged for 5 h at 22,000 rpm in an AH627 rotor. Fractions
containing synaptic vesicle marker proteins were pooled, pelleted by
centrifugation at 48,000 rpm for 2 h, resuspended in buffer A and
then loaded onto an equilibrium density sucrose gradient (10-50%
(w/v) in buffer A) and centrifuged at 48,000 rpm for 18 h in a
SW50.1 rotor. For velocity centrifugation, material from the
equilibrium density gradient was further fractionated in 10-30% (w/w)
gradients in a SW50.1 rotor at 48,000 rpm for 55 min. Following each
centrifugation, fractions were collected starting from the bottom of
the gradient and analyzed for the total protein content, presence of
specific proteins by Western blotting, and aminopeptidase activity.
Purification of Clathrin-coated Vesicles from Rat
Brain--
Clathrin-coated vesicles were purified from rat brain in
MES1 buffer (0.1 M MES, 1 mM EGTA, 0.5 mM
MgCl2, pH 6.5) by the procedure of Maycox et al.
(11). The final pellet containing purified coated vesicles was
resuspended in 0.2 ml of MES buffer, and coated vesicles were then
centrifuged in an equilibrium density sucrose gradient (20-65% (w/v)
in MES buffer, pH 6.5) at 48, 000 rpm for 18 h in a SW50.1 rotor.
Fractions were collected from the bottom of the gradient and analyzed
for total protein and for specific proteins by Western blotting.
Fractions that were positive for clathrin as well as for the synaptic
vesicle proteins SV2, synaptophysin, and synaptobrevin were further
purified by recentrifugation in an additional equilibrium sucrose
gradient (20-65% w/v) for 18 h.
Anion Exchange Chromatography--
To separate proteins by anion
exchange chromatography, membrane samples were solubilized in 1%
Triton X-100 for at least 2 h at 4 °C, centrifuged, and applied
to a 1-ml Pharmacia Mono-Q column equilibrated with buffer A with 50 mM NaCl and 0.1% Triton X-100. Elution was carried out
with a linear gradient of NaCl (final concentration 0.5 M,
total volume of the gradient 30 ml) at a flow rate of 0.5 ml/min. Fifty
1-ml fractions were collected for analysis of the total protein
content, aminopeptidase activity and for Western blotting. The
concentration of NaCl in the gradient fractions was re-evaluated with a
digital conductivity meter.
Fractionation of Triton-solubilized Proteins in Sucrose
Gradients--
Protein solutions (up to 1 mg in 0.15-0.20 ml) in 1%
Triton X-100 in buffer A (clarified by centrifugation for 15 min in a Microfuge) was loaded on a 5-20% sucrose gradient (total volume. 4.6 ml) prepared in buffer A with 0.1% Triton X-100 and spun for 16 h
at 33,000 rpm in a SW-50.1 rotor. The gradients were separated into 46 fractions starting from the bottom of the tube for analysis of the
total protein content, aminopeptidase activity, and for Western
blotting. Standard proteins, bovine serum albumin (67 kDa) and yeast
alcohol dehydrogenase (141 kDa), were analyzed in separate tubes.
Aminopeptidase Activity--
Fractions (10 µl) from the
gradient were mixed in the presence of 1% Triton X-100, with 1 mM (final concentration) arginine-
-naphthylamide (0.5 ml) in a total volume of 1.5 ml of buffer A. The mixture was incubated
for 30 min at 37 °C, and fluorescence in the fractions was measured
at 410 nm. The excitation wavelength was 340 nm.
Immunofluorescent Staining--
PC12 cells grown on coverslips
for 2 days were fixed with 4% paraformaldehyde in PBS containing 4%
sucrose for 30 min, then washed with PBS, permeabilized with 0.25%
Triton X-100 for 5 min, blocked with 4% donkey serum, and stained with
affinity purified antibody against Glut3 followed by Cy3-conjugated
donkey anti-rabbit IgG (Jackson ImmunoResearch). Each incubation with
antibody lasted for 60 min at room temperature. SlowFade-Light Antifade
kit (Molecular Probes) was used for mounting cells on slides. Staining
was examined by confocal laser scanning microscopy (Bio-Rad,
Microscope Division; MRC/600).
Electron Microscopy--
Glut3-containing vesicles purified by
sucrose gradient centrifugation were pelleted, resuspended in PBS to a
final concentration 0.1-0.5 mg/ml, and fixed with 2% paraformaldehyde
in PBS. Formvar-carbon coated nickel grids were layed on 25-µl drops
of vesicle suspension for 1-5 min. Grids were then stained with 1%
uranyl acetate for 30 s, dried, and viewed through a Phillips
transmission electron microscope. Micrographs were taken at
magnification × 10,000-48,000.
Gel Electrophoresis and Immunoblotting--
Proteins were
separated by SDS-polyacrylamide gel electrophoresis according to
Laemmli (12) and transferred to an Immobilon-P membrane in 25 mM Tris, 192 mM glycine. Following transfer,
the membrane was blocked with 10% nonfat dry milk in PBS for 1 h
at 37 °C and probed with specific antibodies. Autoradiograms were quantitated in a computing densitometer (Molecular Dynamics).
Protein Determination--
Protein content was determined with
the BCA kit (Pierce) according to the manufacturer's instructions.
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RESULTS |
Identification and Isolation of the Intracellular Glut3-containing
Vesicles--
Fig. 1 shows localization
of Glut3 in the neuroendocrine PC12 cell line by immunofluorescent
staining. In this figure, it is evident that in addition to the plasma
membrane, Glut3 is present in an intracellular perinuclear
compartment.

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Fig. 1.
Localization of Glut3 in PC12 cells. The
plane of focus at an intermediate section through the cells shows
localization of Glut3 in the plasma membrane and perinuclear region of
PC12 cell.
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To understand the nature of this intracellular Glut3-containing
compartment, we fractionated PC12 cells as described previously (9).
After cell homogenization, heavy subcellular structures and the plasma
membrane were removed by pelleting, and the resulting intracellular
membranes were fractionated in a linear equilibrium density gradient as
described under "Materials and Methods." Under these conditions,
Glut3-containing intracellular membranes have a very narrow density
distribution that substantially, although not completely, overlaps with
the position of the synaptic vesicle marker proteins SV2 and
synaptophysin, with Glut3-containing vesicles having a buoyant density
less than synaptic vesicles (Fig. 2).

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Fig. 2.
Fractionation of synaptic vesicles from PC12
cells in an equilibrium density gradient. Synaptic vesicles were
partially purified from PC12 cells as described under "Materials and
Methods," reapplied on a 10-50% (w/v) sucrose equilibrium density
gradient, and centrifuged for 18 h at 48,000 rpm in an SW50.1
rotor. Fractions were collected starting from the bottom of the
gradient and blotted for GLUT3 ( ), SV2 ( ), and synaptophysin
( ). Autoradiograms were quantitated in a computing densitometer
(Molecular Dynamics). The figure shows a representative result of six
independent experiments.
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The limited amount of the material available from cultured cells did
not allow us to purify intracellular Glut3-containing vesicles from
PC12 cells in the preparative amounts that were required for detailed
biochemical analysis of this compartment. Therefore, further
characterization of Glut3-containing vesicles was performed using rat
brain as a source of biological material.
In the following experiments, we took advantage of a well established
procedure for the isolation and subcellular fractionation of
synaptosomes (10). Briefly, we isolated synaptosomes from rat brain,
lysed them with osmotic shock, removed synaptic membranes, and purified
intra-synaptosomal membranes by preparative sucrose gradient
centrifugation (10). This preparation was fractionated further by
equilibrium density sucrose gradient centrifugation as shown in Fig.
3 and as described previously (7). This
procedure allows the separation of the intra-synaptosomal membrane
material into two major peaks according to their buoyant densities (7), one of which is likely to represent classical synaptic vesicles (7).
The nature of the second less dense population of vesicles remains
unknown. Fig. 3 shows that Glut3 is specifically enriched in this
second population of vesicles. Based on these results, we conclude that
the buoyant density distribution of Glut3 vesicles and synaptic
vesicles in PC12 cells (Fig. 2) and rat brain (Fig. 3) is very similar
if not identical.

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Fig. 3.
Fractionation of synaptic vesicles from rat
brain in an equilibrium density sucrose gradient. Partially
purified synaptic vesicles were loaded on an equilibrium density
sucrose gradient (10-50%). Fractions were analyzed for total protein
( ) and aminopeptidase activity ( ). Equal volume aliquots from
fractions of the equilibrium density gradient were subjected to
SDS-polyacrylamide gel electrophoresis and individual proteins were
analyzed by Western blotting. The figure shows a representative result
of at least fifteen independent experiments.
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As we have shown earlier (7) and reconfirm here, this second population
of "synaptic-like" vesicles from rat brain, unlike classical
synaptic vesicles, possesses a high level of aminopeptidase activity
(Fig. 3), which serves as a convenient and reliable marker of this
compartment and allows one to readily distinguish the two populations
of vesicles.
The peak 2 fractions, which contained both Glut3 and
aminopeptidase, were pooled and, following pelleting, recentrifuged
in a sucrose velocity gradient (Fig. 4).
Under these conditions, the Glut3-containing material sediments as
homogeneous vesicles whose distribution completely overlaps with the
total protein profile and also with synaptic vesicle marker proteins
and aminopeptidase activity.

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Fig. 4.
Fractionation of Glut3-containing vesicles in
a sucrose velocity gradient. Glut3- and aminopeptidase-containing
fractions of the equilibrium density gradient (peak 2 in Fig. 3) were
pelleted, resuspended, and centrifuged in a 10-30% sucrose velocity
gradient. Fractions were analyzed for total protein ( ) and odd
fractions only, for aminopeptidase activity ( ). Equal volume
aliquots of even fractions were subjected to SDS-polyacrylamide gel
electrophoresis, and individual proteins were analyzed by Western
blotting. The figure shows a representative result of at least five
independent experiments.
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Data presented in Fig. 4 suggest that Glut3-containing vesicles at this
purification step are close to homogeneity. This was confirmed by
electron microscopy (Fig. 5), which
demonstrated that this preparation consists of homogenous round-shaped
vesicles with an average diameter of 47 ± 5 nm. Using electron
microscopy, we could not find any significant differences between
Glut3-containing synaptic-like vesicles and classical synaptic
vesicles (not shown).

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Fig. 5.
Electron microscopy of purified
Glut3-containing vesicles. Purified Glut3-containing vesicles
(Fig. 4) were pooled and analyzed by transmission electron microscopy
as described under "Materials and Methods." Bar, 100 nm.
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Identification of the Aminopeptidase Activity Associated with
Glut3-containing Vesicles as Aminopeptidase B--
To identify
aminopeptidase activity associated with Glut3-vesicles, we used an
antibody against aminopeptidase B described by Cadel et al.
(13). For these experiments, we solubilized Glut3-containing vesicles
in 1% Triton X-100 and centrifuged this material in a 5-20% linear
sucrose gradient. Under these conditions, aminopeptidase activity
sediments as one peak corresponding to a protein of ~70 kDa and
completely overlaps with the distribution of aminopeptidase B as
determined by Western blotting (Fig.
6).

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Fig. 6.
Fractionation of Triton-solubilized proteins
of Glut3-containing vesicles in sucrose gradient. Glut3-containing
vesicles purified by sucrose gradient centrifugation were solubilized
in 1% Triton X-100 (total volume 0.2 ml) and fractionated in a 5-20%
sucrose gradient as described under "Materials and Methods."
Fractions were analyzed for total protein content ( ), aminopeptidase
activity ( ) (top panel) and for aminopeptidase B by
Western blotting (bottom panel). The positions of bovine
serum albumin (67 kDa) and yeast alcohol dehydrogenase (141 kDa) are
indicated by vertical arrows. The figure shows a
representative result of three independent experiments.
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In the next experiment, we fractionated Triton-solubilized
Glut3-containing vesicles on a Mono-Q column (Fig.
7). Unlike sucrose gradient
centrifugation, fast protein liquid chromatography allows the
separation of aminopeptidase activity into two peaks, the major of
which co-elutes with aminopeptidase B. The nature of the minor peak
remains unknown, although it may represent a degradation product of the
major peak or may result from binding of aminopeptidase B to other
proteins. Thus, we conclude that aminopeptidase B is the only or, at
least, the major aminopeptidase present in Glut3-containing vesicles.
Because aminopeptidase B has recently been shown to be targeted to the
regulated secretory pathway in PC12 cells (8), we believe that
Glut3-containing vesicles, which we describe here, represent a
secretory compartment in both PC12 cells and neurons.

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Fig. 7.
Fractionation of Triton-solubilized proteins
of Glut3-containing vesicles on a Mono-Q column. Glut3-containing
vesicles purified by sucrose gradient centrifugation were solubilized
in 1% Triton X-100 (total volume 2 ml), applied to a Mono-Q column
(Amersham Pharmacia Biotech), and washed with 18 ml of buffer A with 50 mM NaCl and 0.1% Triton X-100. The gradient of NaCl
(50-500 mM) starts at fraction 20 and increases linearly
until it reaches fraction 50. All fractions were analyzed for total
protein content ( ), aminopeptidase activity ( ) (top
panel), and for aminopeptidase B by Western blotting (bottom
panel). The figure shows a representative result of three
independent experiments.
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Glut3 in Clathrin-coated Vesicles--
Glucose transporter
proteins, Glut1, and Glut4, in particular, are known to recycle between
the plasma membrane and an intracellular storage compartment. To
determine whether this may also be the case for Glut3, we isolated
clathrin-coated vesicles from rat brain according to the procedure of
Maycox et al. (11) and fractionated them in an equilibrium
density sucrose gradient (Fig. 8). Glut3 is clearly present in clathrin-coated vesicles suggesting that this
isoform of glucose transporter recycles in neurons, although the
quantitative parameters of this recycling have yet to be
determined.

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Fig. 8.
Isolation of clathrin-coated vesicles in a
sucrose equilibrium density gradient. Clathrin-coated vesicles
were purified as described under "Materials and Methods." Fractions
positive for clathrin and synaptic vesicle proteins were pooled,
pelleted, and recentrifuged in a 20-65% sucrose equilibrium density
gradient. This figure demonstrates total protein profile and Western
blot analysis of equal volume aliquots of every even fraction. A
representative result of three independent experiments is shown.
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DISCUSSION |
In this study, we examine the intracellular localization of Glut3,
the predominant glucose transporter isoform in brain. We show that
Glut3 is present in a distinct homogenous population of synaptic-like
vesicles that we have recently identified in rat brain (7) where these
vesicles are as abundant as classical synaptic vesicles and can be
separated from the latter by equilibrium density gradient
centrifugations. The overall protein composition of these synaptic-like
vesicles is very close to classical synaptic vesicles, with SV2,
synaptotagmin, synaptophysin, and synaptobrevin being the major
components in both pools (7). Several important differences have
nevertheless been found in protein content in the two vesicular
populations. First, synaptic-like vesicles have a much lower content of
V-type H+-ATPase (proton pump) (7); second, they
specifically compartmentalize Glut3 (this study); finally, they have
very high aminopeptidase activity that has been identified as
aminopeptidase B (this study). Because this aminopeptidase is known to
be targeted to the secretory pathway (8), we believe that these
synaptic-like vesicles represent a secretory compartment in neurons and
PC12 cells. This is consistent with the fact that Glut3 is targeted to
the secretory granules in platelets, another cell type with high Glut3
content (4, 5). Because Glut3 is also a predominant component of
clathrin-coated vesicles in rat brain, we suggest that this protein may
recycle between its intracellular compartment and the plasma membrane via an as yet uncharacterized pathway involving
clathrin-coated vesicles.
There are interesting parallels between Glut3-containing vesicles in
brain and Glut4-containing vesicles in insulin-sensitive fat and
skeletal muscle tissues. Both types of vesicles have similar size,
sedimentation coefficient, and buoyant density in sucrose solutions
(this study and Ref. 14). Moreover, both incorporate aminopeptidases
that, in addition to glucose transporters, serve as marker proteins for
these vesicular compartments. The specific vesicle-associated
aminopeptidases, however, are quite different; insulin-sensitive
aminopeptidase, or IRAP, was found in Glut4-containing vesicles (15,
16) and aminopeptidase B (8, 17) in Glut3-containing vesicles. An even
more significant difference is that Glut4-containing vesicles represent
a specialized endosomal compartment in adipocytes (18), whereas
Glut3-containing vesicles in brain are likely to be a regulated
secretory compartment. In this regard, it will be interesting to
determine whether Glut3- and aminopeptidase B-containing synaptic-like
vesicles are relevant to the vesicular compartment identified by
transfection of Glut4 into PC12 cells (19). This may reveal interesting
aspects of protein targeting into different specialized intracellular
membrane compartments. Another important question has to do with the
regulation of Glut3 translocation from intracellular secretory vesicles
to the cell surface and its possible return to an intracellular pool,
which presumably takes place via a clathrin-mediated
mechanism. This problem is currently under investigation in our laboratory.