From the Centro de Biología Molecular Severo Ochoa. CSIC. Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049, Spain
Received for publication, July 27, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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The insulin-sensitive glucose transporter GLUT4
is translocated to the plasma membrane in response to insulin and
recycled back to the intracellular store(s) after removal of the
hormone. We have used clonal 3T3-L1 fibroblasts and
adipocyte-like cells stably expressing wild-type GLUT4 to
characterize (a) the intracellular compartment where the
bulk of GLUT4 is intracellularly stored and (b) the
mechanisms involved in the recycling of endocytosed GLUT4 to the store
compartment. Surface internalized GLUT4 is targeted to a large, flat,
fenestrated saccular structure resistant to brefeldin A that
localized to the vicinity of the Golgi complex is sealed to endocytosed
transferrin (GLUT4 storage compartment). Recycling of endocytosed GLUT4
was studied by comparing the cellular distributions of antibody/biotin
tagged GLUT4 and GLUT4(Ser5), a mutant with the
Phe5-Gln6-Gln7-Ile8
inactivated by the substitution of Ser for Phe5. Ablation
of the
Phe5-Gln6-Gln7-Ile8
inhibits the recycling of endocytosed GLUT4 to the GLUT4 store compartment and results in its transport to late endosomes/lysosomes where it is rapidly degraded.
Plasma euglycemia is maintained by the effect of insulin on muscle
and to a lesser extent on adipose tissue. In these tissues insulin
stimulates glucose transport, the rate-limiting event in glucose
disposal (1-3). The function of GLUT4, the only glucose transporter
sensitive to insulin, is essential to maintain the insulin-regulated
plasma euglycemia, which is controlled through the regulation of the
GLUT4 trafficking. Insulin stimulates glucose transport by promoting
the translocation of GLUT4 (4, 5) from the intracellular
tubulovesicular structures where it is stored (6-8) to the plasma
membrane (5, 7, 9, 10). After withdrawal of the hormone, GLUT4 is
removed from the plasma membrane and recycled to the intracellular
stores, and the steady-state previous to insulin stimulation is
re-established (11, 12).
The molecular mechanisms involved in GLUT4 trafficking are poorly
understood. The 12 transmembrane domain protein displays, in its amino
and carboxy-cytoplasmic tails,
Phe5-Gln6-Gln7-Ile8-based
(13) and Leu489-Leu490-based motifs (14) that
when inactivated provoke its cellular redistribution. Both motifs have
been involved in GLUT4 endocytosis, intracellular retention, and
targeting in transfected 3T3-L1fibroblasts, COS-7 and Chinese hamster
ovary cells (15-20), myoblasts (21), and adipocytes (18, 22-24).
Whether the dileucine motif mediates the trafficking of GLUT4 in
adipocytes is, however, the subject of much debate (18, 22, 23). In
addition, the mechanisms that mediate the intracellular retention and
recycling of GLUT4 to the intracellular store compartments remain
essentially unknown.
Here we have investigated the motifs involved in the recycling of
endocytosed GLUT4 to the GSC1
and study the organization and localization of this. For this purpose,
we have stably expressed HA epitope-tagged wild-type GLUT4 and a
GLUT4(Ser5), a mutant with the
Phe5-Gln6-Gln7-Ile8
motif ablated, and studied the intracellular distribution of endocytosed molecules tagged with anti-HA antibodies and the
GLUT4-specific reagent Bio-LC-ATB-BMPA. The results of these studies
show that the
Phe5-Gln6-Gln7-Ile8
motif is critical for access of endocytosed GLUT4 to the GSC where the
bulk of it is stored. The GSC consists of a flat saccular structure
resistant to BFA organized as a reticulum at one of the nuclear poles.
Cell Culture--
3T3-L1 fibroblasts were cultured on plastic
dishes or glass coverslips in DMEM supplemented with 10% fetal calf
serum, 4 mM glutamine, 50 mg/liter streptomycin, 100 IU/liter penicillin, nonessential amino acids (complete medium) in a
37 °C humidified CO2 incubator.
Differentiation of 3T3-L1 Fibroblasts into Adipocyte-like
Cells--
Clonal 3T3-L1 fibroblasts kept confluent for 3 days were
incubated for 3-7 days in IDBX medium (1.6 × 10 Antibodies--
The mouse monoclonal (mAb16B12) and the rabbit
polyclonal (Y11) anti-HA antibodies were from BabCO (Berkeley, CA) and
Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The rabbit polyclonal anti-exofacial loop antibody (pAbEL) was raised against a
mixture of two peptides (NH2-CRQGPGGPDSIPQGTLTTLWA-COOH and NH2-CNAPQKVIEQSYNATWLC-COOH) that stretch out from residue
44 to residue 83 on the first large exofacial loop of GLUT4. The rabbit
pAb828 was raised against the peptide representing the COOH-tail of
GLUT4. These four antibodies were used to study the distribution of
native and tagged GLUT4 molecules by IMF microscopy, electron
microscopy, and Western analysis. Their use was conditioned by
the protocols employed and the origin of the antibodies used as
organelle markers (see below). Occasionally the experiments were
repeated with two different anti-GLUT4 antibodies to evaluate their
effects on the system under study. pAb828 was covalently bound to
CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) and used to
study the fraction of pAbEL bound to the GLUT4 and GLUT4(Ser5) molecules recovered with purified LDM
fractions. pAbEL was conjugated to FITC to compare the distribution of
surface internalized tagged GLUT4 or GLUT4(Ser5) with Texas
red-Tfn and Texas red-dextran beads. The mouse anti-His mAb, rabbit
anti-Rab5b, and anti-Rab4 pAb were from Santa Cruz Biotechnology (Santa
Cruz, CA). The development of the rabbit pAb35C8 against LIMPIII, a
membrane protein marker of late endosome/lysosome, has been reported
(25). The rabbit pAb against the Golgi sialoglycoprotein MG160 was a
gift of Dr. Gonatas (26). The rabbit pAb18B11 against the
trans-Golgi network marker GMPt DNA Constructs--
The cDNAs of GLUT4 were cloned into the
modified pPUR vector (CLONTECH) carrying the spleen
focus forming virus (SFFV) promoter at the
ApaI/EcoRI sites (20). GLUT4 was tagged by
introducing the HA epitope after Thr78 into the GLUT4
cDNA carried in the pPUR vector (20, 28). Ser was replaced for
Phe5 by site-directed mutagenesis using as template
HA-GLUT4 (29) (see Fig. 1A). To introduce the His tag at the
COOH-end of HA-GLUT4, this was cloned into the
EpiTagTM/Myc-His vector (Invitrogen, NL).
Development of Clonal Stable Transfectants--
3T3-L1
fibroblasts were transfected with cDNAs cloned into the pPUR vector
using the calcium phosphate precipitation method (30, 31). Stably
transfectants were selected with 7.5 µg/ml puromycin and screened for
clones homogeneously expressing the transfected proteins first by IMF
microscopy. Developed clones were grown in complete medium and
maintained in 7.5 µg/ml puromycin.
Sensitivity of GLUT4/GLUT4(Ser5)-Antibody Complexes
to Low pH--
To study the stability of [35S]GLUT4 and
[35S]GLUT4(Ser5)/antibody complexes within
the pH 5.5-7.4 range, purified protein G-Sepharose complexes were
incubated for 10 min or 1 h at 37 °C with either 50 mM Tris-HCl, pH 7.4, or 50 mM cacodylate
buffer, pH 6.4 or 5.5, containing100 mM NaCl and 2 mM EDTA. Intact and dissociated complexes were separated by
low speed centrifugation and resolved by SDS-PAGE, and their levels
were measured by autoradiography.
Studies on the Cellular Levels and Turnovers of GLUT4
Species--
Cell membranes from 3T3-L1 fibroblasts were prepared as
follows. Cells were washed twice with phosphate-buffered saline,
scrapped, and incubated for 30 min in 2 ml of 0.1 M
Na2CO3, pH 11.3, and membrane pellets were
prepared by centrifugation at 150,000 × g for 30 min.
The membranes were then solubilized for 30 min with 50 mM
Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100 (buffer A) and, after removal of the insoluble material by centrifugation at
150,000 × g for 30 min, diluted in 1 volume of buffer
A without detergent. All the manipulations were performed at
4 °C.
The cellular levels of GLUT4 and GLUT4(Ser5) mutant were
measured in 30 µg of membrane protein by Western analysis using the pAb828 and the ECL technique (Amersham Pharmacia Biotech). To study the
fraction of GLUT4 exposed on the surface of stably transfected clonal
3T3-L1 fibroblasts and ADL cells, cells grown to 90% confluence on
60-mm plastic dishes were incubated for 20 min at 37 °C with 2 mM KCN prepared in Krebs-Ringer-Hepes buffer prior
incubation for 10 min at 37°C with or without 2.5 mg/ml trypsin
(Sigma) (28). After stopping the trypsin digestion by addition of 0.1 M phenylmethylsulfonyl fluoride and 2 mg/ml of soybean
inhibitor (Sigma), the cells were harvested by centrifugation at low
speed and 30 µg of membrane protein, prepared as described above, and
analyzed by Western blot using the anti-GLUT4 pAb828. It should be
noted that the introduction of the HA tag into the large exofacial loop
of GLUT4 enhanced the sensitivity of the GLUT4 molecules to trypsin.
The turnover of GLUT4 and GLUT4(Ser5) were studied in
3T3-L1 fibroblasts incubated for 30 min in methionine/cysteine-free
complete medium and then labeled for 10 min or 1 h with
[35S]methionine/cysteine (>1,000 Ci/mmol) (Amersham
Pharmacia Biotech) before their chase for different time periods with
cold complete medium (25). To study the effect of the inhibition of
lysosomal proteases on their turnover, the cells were preincubated for
1 h with 50 mM chloroquine and 100 µg/ml leupeptine,
and then the inhibitors were added to the labeling and chase
medium. GLUT4 and GLUT4(Ser5) were
immunoprecipitated using the mAb16B12 and pAbEL bound to protein G
-Sepharose (25) (Amersham Pharmacia Biotech) and, after their
resolution by 10% PAGE, quantitated by autoradiography.
Uptake and Distribution of Tagged GLUT4 Molecules--
Clone
3T3-L1 fibroblasts and ADL cells stably expressing GLUT4 or the
GLUT4(Ser5) mutant were incubated for time periods of 15 min, 1 h, and 18 h with 50 µg/ml of either mAb16B12 or
pAbEL prepared in complete medium and, when required, washed and
further incubated for different time periods in antibody-free complete
medium. To biotinylate the molecules of GLUT4 and
GLUT4(Ser5) exposed onto the cell surface, 3T3-L1
fibroblasts stably expressing the proteins and grown on 10-mm glass
coverslips were incubated with 1 mM Bio-LC-ATB-BMPA. (a
gift of Dr. Geoff Holman, University of Bath, Bath, UK) prepared in 30 µl of Krebs-Ringer-Hepes buffer prepared (32) and UV irradiated six
times for 1 min at intervals of 5 min.
To investigate whether the GSC was a compartment distinct from the
perinuclear recycling compartment traversed by Tfn, fibroblasts stably
transfected with GLUT4 or GLUT4(Ser5) were preincubated for
1 h at 37 °C with FITC-pAbEL, washed, incubated for 10 min with
Texas red-Tfn (50 µg/ml; Molecular Probes, Eugene, OR), and then
incubated for 10-50 min in plain complete medium. To study whether the
endocytosed antibody-tagged GLUT4(Ser5) was downloaded into
late endosomes/lysosomes, fibroblasts stably transfected with
GLUT4(Ser5) were preincubated for 1 h at 37 °C with
FITC-pAbEL and then incubated for 10 min with Texas red-dextran beads
(molecular weight, 70,000; at 0.1 mg/ml; Molecular Probes), before
their wash and incubation for 50 min in plain complete medium.
Processing of the cells for IMF microscopy was as follows. The cells
were quickly washed three times with phosphate-buffered saline and then
fixed with 4% paraformaldehyde, permeabilized with cold methanol and
stained with specific antibodies or streptavidin (25). FITC and Texas
red-conjugated antibodies were from Cappel (Durham, NC).
FITC-conjugated streptavidin was from Jackson Immunoresearch (West
Grove, PA). The cellular distributions of tagged GLUT4 molecules, Tfn,
and dextran beads were studied by IMF microscopy using an Axiovert 135 M inverted microscope (Zeiss) and a confocal Radiance 2000 microscope (Bio-Rad). The cells were photographed through fluorescence.
Electron Microscopy Studies--
3T3-L1 fibroblasts stably
expressing GLUT4 and incubated with pAbEL were processed for electron
microscopy studies using the pre-embedding procedure (25).
Endogenous peroxidase was blocked by incubating the fixed/permeabilized
cells for 30 min at room temperature with 0.4%
H2O2 before their incubation with a
HRP-conjugated F(ab')2 donkey anti-rabbit antibody.
Subcellular Fractionation Studies--
The purification of high
density (HDM) and low density microsomes (LDM) from 3T3-L1 fibroblasts
and ADL cells was performed as described (33). LDM were fractionated by
centrifugation on sucrose velocity gradients by a modification of a
procedure described recently (34). Before harvesting, cells grown for
72 h on 10-cm dishes were poisoned for 5 min at room temperature
with 2 mM KCN and then quickly washed twice with cold
phosphate-buffered saline. Harvesting was in 2 ml of buffer 20 mM Hepes, 250 mM sucrose, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.4 (buffer B). All the subsequent manipulations were performed at 4 °C. Cells were disrupted with 30 strokes using a Potter Evehjem homogenizer (0.08 mm clearance), and the resulting homogenate was centrifuged at
16,000 × g for 20 min. HDM were pelleted from the
resulting supernatant by centrifugation for 30 min at 30,000 × gmax, and LDM collected form the resulting
supernatant by centrifugation at 150,000 × gmax for 90 min. LDM vesicles were resuspended
in 400 µl of buffer B, loaded onto a 4.6 ml of 12-30% sucrose
gradient prepared in 20 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, and fractionated by
centrifugation at 300,000 × gmax for 55 min using a SW50.1 rotor (Beckman Instruments, Palo Alto, CA).
Fractions were collected from the top of the gradient. The refractive
index of the collected fractions was measured to assess the linearity
of the gradients. The gradient profiles of GLUT4,
GLUT4(Ser5), Rab4, Rab5b, and endocytosed pAbEL were
studied by Western analysis using specific antibodies and the ECL technique.
To characterize the intracellular GSC and to study the role of the
Phe5-Gln6-Gln7-Ile8
motif in the recycling of GLUT4 from the cell surface to the GSC, we
traced the intracellular distribution of endocytosed antibody/biotin tagged GLUT4 and GLUT4(Ser5) molecules by IMF microscopy,
electron microscopy, and Western analysis.
Cellular and Plasma Membrane Levels of GLUT4 and
GLUT4(Ser5) in 3T3-L1 Fibroblasts and ADL
Cells--
3T3-L1 clones transfected with a modified pPUR plasmid (20)
carrying the GLUT4 and GLUT4(Ser5) cDNAs (Fig.
1A) and selected for their
ability to grow in puromycin were scrutinized for the capacity of their
cells to express the transfected proteins by IMF microscopy using
pAb828. Only clones homogeneously expressing the transfected proteins
were selected for further scrutiny of their cellular levels.
The cellular levels of transfected GLUT4 and GLUT4(Ser5)
were measured in clonal 3T3-L1 fibroblasts to select clones with
comparable levels of the two proteins and to discard those expressing
high levels that could saturate the mechanisms of GLUT4 transport. For
this purpose, whole membranes were prepared from clones selected by IMF
microscopy, and their content in GLUT4 and GLUT4(Ser5) was
studied by Western analysis using pAb828 and the ECL technique (Fig.
1C). The levels of the wild-type protein and the mutant were
also analyzed in ADL cells differentiated from fibroblasts in
vitro and used before the expression of endogenous GLUT4 (day 2 of
insulin treatment; Fig. 1B). Their study revealed that their levels remained comparable after differentiation (Fig.
1D).
The levels of GLUT4 and GLUT4(Ser5) in the plasma membrane
of 3T3-L1 fibroblasts and ADL cells were also compared using a trypsin protection assay. For this purpose, cells poisoned with CNBr were incubated for 10 min at 37 °C with 2.5 mg/ml trypsin (28), and the
digestion of GLUT4 and GLUT4(Ser5) was studied by Western
analysis using pAb828 and the ECL technique. The study showed that, in
fibroblasts, the surface levels of GLUT4(Ser5), 22 ± 2%, were significantly higher than those of GLUT4, 5.5 ± 1%
(Fig. 1B). Comparable results were obtained in ADL cells as
the percentage of GLUT4(Ser5) and GLUT4 were 38 ± 2 and 6.5 ± 0.5% (Fig. 1D). Furthermore, when ADL cells
were preincubated for 2 h with DMEM and then for 40 min with
10 GLUT4-Antibody Complexes are Highly Stable at Acidic
pH--
Actively endocytosed antibodies when transported through
endosomes are exposed to acidic environments that may provoke their dissociation from bound antigens (35). The use of antibodies to study
the targeting of GLUT4 from the cell surface to the GSC required,
therefore, to demonstrate that HA-tagged GLUT4/antibody complexes
remained stable at low pH. For this purpose, [35S]GLUT4
and [35S]GLUT4(Ser5)/antibody complexes,
purified from metabolically labeled cell extracts with mAb16B12/protein
G-Sepharose (Fig. 2A), were
incubated for 10 min or 1 h at 37 °C at pH 7.4, 6.4, and 5.5, and the effect of pH on their stability was studied by measuring GLUT4
and GLUT4(Ser5) in the supernatants and pellets separated
by low speed centrifugation. The results showed that GLUT4 (upper
panel) and GLUT4(Ser5) (lower panel)
remained bound to the antibody even at pH 5.5, a value lower than the
pH measured in endosomes and typical of lysosomes (35). Comparable
results were obtained when the study was repeated using pAbEL (not
shown). From these results as well as from the results of experiments
performed in vivo (see Fig. 10), we concluded that mAb16B12
and pAbEL could be used to study the recycling of GLUT4 from the cell
surface to the GSC.
Antibody-tagged GLUT4 Accumulates in LDM Vesicles in a Manner
Regulated by the
Phe5-Gln6-Gln7-Ile8
Motif--
To begin to characterize the pathway of GLUT4 recycling
from the cell surface to the GSC, clonal 3T3-L1 fibroblasts either untransfected or stably expressing GLUT4 or GLUT4(Ser5)
were incubated for 1 h at 37 °C with mAb16B12 (Fig.
2B) or pAbEL (data not shown), and the antibody recovered
with the fractions enriched in LDM and HDM was studied by Western
analysis using the ECL technique. The results showed that fibroblasts
stably expressing GLUT4 preferentially accumulated the endocytosed
antibody in LDM as compared with HDM (Fig. 2B). Moreover,
ablation of the Phe5-Gln6-Gln7-Ile8
motif dramatically decreased the accumulation of the antibody in LDM
vesicles (Fig. 2B). Comparable results were obtained when the studies were repeated in ADL cells (not shown).
Surface Internalized GLUT4 Is Targeted to the GSC--
3T3-L1
fibroblasts stably expressing HA-GLUT4 immunostained with anti-GLUT4
pAb828 concentrated GLUT4 in a perinuclear structure organized as a
reticulum at one of the nuclear poles, and the rest was in punctuate
structures scattered throughout the cytoplasm (Fig.
3A). The perinuclear
compartment will be referred henceforth as the GSC, for GLUT4 storage
compartment (20).
To investigate the recycling of GLUT4 from the cell surface to the GSC,
fibroblasts stably transfected with GLUT4 were incubated for periods of
15 min, 1 h, and 18 h at 37 °C with 50 µg/ml of mAb16B12
or were UV irradiated in Krebs-Ringer-Hepes buffer containing the GLUT4
affinity reagent, Bio-LC-ATB-BMPA. After treatment, the cells were
washed and further incubated at 37 °C for 1 h in plain complete
medium or were immediately processed for microscopy. The distribution
of the tagged GLUT4 molecules was studied after cell fixation with 4%
paraformaldehyde and permeabilization with methanol. mAb16B12 was
stained using FITC/HRP-conjugated goat anti-mouse antibodies and
Bio-LC-GLUT4 with FITC-streptavidin. The results with the two tags were
comparable (Fig. 3, B and C). The bulk of
mAb16B12 internalized by cells incubated for 1 h with the antibody
and then for 1 h in plain complete medium was localized to a
reticular structure localized to one of the poles of the nucleus, both
organization and localization characteristic of the GSC (Fig. 3,
compare A, B, and C). Comparable
results were obtained when the incubation with the antibody was reduced
to 15 min or extended to 18 h (data not shown). When after
incubation with the antibody the cells were immediately processed, the
antibody was localized, in addition to the perinuclear reticular
structure, to numerous punctate structures preferentially clustered in
the same perinuclear area (not shown). To further investigate the similarities between the compartment that retained the internalized antibody and the GSC, cells incubated for 1 h with mAb16B12 were double-stained for the antibody and the Golgi sialoglycoprotein MG160
(26), and their distribution was compared by IMF microscopy.
The study, performed in flat spreaded fibroblasts, showed that the
compartment loaded with mAb16B12 was closely juxtaposed to the Golgi
complex (Fig. 3D), again a main characteristic of the GSC
(20). Moreover, when in the same experiment the distributions of the
internalized antibody and the TGN marker GMPt
The vacuolar compartments beyond the trans-Golgi have been
shown to respond to BFA with the production of stable tubular
expansions, a phenomenon in contrast with the short life expansions
produced by the Golgi complex (36-38). To further characterize the GSC
and to establish its localization with relation to the Golgi,
fibroblasts stably expressing GLUT4 and preincubated for 1 h with
mAb16B12 were further treated for 1 h with 10 mg/ml BFA and then
stained for the antibody and GLUT4. In parallel, fibroblasts similarly treated were incubated for 10 min or 1 h with the drug and the Golgi stained with the anti-MG160 antibody to monitor the Golgi response to the drug. The study of the mAb16B12 distribution showed that BFA induced the production of highly stable tubular expansions from the GSC, but despite these morphological changes after 1 h of
drug treatment, the GSC remained as a distinct structure in the
vicinity of the nucleus (Fig. 3, F and G). This
stability was in contrast with the rapid disassembly of the Golgi
complex, which disappeared after 10-20 min of incubation with the drug (Fig. 3, compare F and G with H and
I). This different response, while indicating differences in
the flow of membranes that traverse the Golgi and GSC, strongly
suggests that the GSC is organized as a distinct structure beyond the
TGN. It is important that comparable results were obtained when the
same experiments were repeated with cells incubated with pAbEL (Fig.
5).
The GSC Is a Compartment Distinct from the Recycling Compartment
Traversed by Transferrin--
Molecules internalized by endocytosis
and recycled back to the cell surface as Tfn often move through a
recycling compartment localized to the vicinity of the nucleus (39).
Because of their similar localization, it was important to investigate
whether the GSC and the endocytic recycling compartment were distinct structures. For this purpose, 3T3-L1 fibroblasts stably expressing GLUT4 and preincubated for 1 h with FITC-pAbEL were then incubated for 10 min with Texas red-Tfn and, after their wash, further incubated for 10 or 50 min in plain complete medium. All the incubations were
done at 37 °C (Fig. 5). The cellular distributions of pAbEL and
transferrin were studied by confocal microscopy. It was observed that
10 min after the Tfn loading, the endocytosed antibody and Tfn
colocalized in numerous punctuate structures. In addition, the antibody
was localized to a reticulum with the morphology and localization
characteristic of the GSC. More important, Tfn was completely excluded
from the reticulum (Figs. 5, A-D). Incubations of up to 90 min in plain complete medium resulted in a decrease of the number of
punctate structures loaded with antibody and transferrin, whereas the
bulk of the endocytosed antibody remained in the GSC (Fig. 5, compare
E and F). These distribution profiles strongly
suggested that the endocytosed antibody and Tfn were transported to the
perinuclear area packed in the same vesicles, a result consistent with
previous observations (40-42), where they were sorted and the antibody
was specifically targeted to the GSC. Furthermore, the exclusion of Tfn
from the GSC clearly showed that the GSC and the recycling compartment
were distinct structures.
GSC Ultrastructure--
To study the ultrastructure of the
organelles involved in the recycling of GLUT4, 3T3-L1 fibroblasts
stably expressing GLUT4 and incubated for 1 h with pAbEL were
studied by electron microscopy, using the pre-embedding
technique. The endocytosed antibody was localized to numerous
tubulovesicular structures, the majority cramming the perinuclear area
(Fig. 6, A, C,
D, E, and H), as well as to
multivesicular bodies (not shown). Occasionally, the antibody was found
in large polymorph structures (Fig. 6F). The en face view of
these large structures revealed their saccular morphology and the
existence of multiple fenestration in their walls (Fig. 6G).
This morphology was reminiscent of the Golgi cisternae, described as
large, flat sheets with a variable degree of fenestration (43). Because
of the large size of these saccular structures, it is likely that their
sectioning during the processing of the cells could account for some of
the tubules and vesicles counted in the perinuclear area.
Endocytosed GLUT4 Is Targeted to the GSC in ADL
Cells--
Adipocytes are together with muscle cells (4) and a few
distinct neurons (44-49) the only cells expressing GLUT4 in
vivo. It was important, therefore, to study the distribution of
endogenous GLUT4 in ADL cells and then to compare this with the
distributions of stably transfected GLUT4 and endocytosed antibody in
the same cells. The distribution of endogenous GLUT4 was studied in ADL cells cultured for 7 days in complete medium supplemented with 10
The recycling of GLUT4 in ADL cells stably transfected with GLUT4 was
studied before the beginning of the endogenous GLUT4 expression (Fig.
1B). The cells, which displayed a cytoplasm crammed with
large lipid droplets and the round morphology characteristic of
adipocytes (Fig. 7, D and E), were incubated at
37 °C for 1 h with mAb16B12 and the distributions of the
endocytosed antibody and GLUT4 studied by IMF microscopy. Again, as in
fibroblasts, the bulk of the antibody was localized to the GSC (Fig. 7,
F and G), thus indicating that in ADL cells the
antibody-tagged GLUT4 was targeted from the cell surface to the
GSC.
Ablation of the
Phe5-Gln6-Gln7-Ile8
Motif Blocks the Targeting of Tagged GLUT4 to the GSC--
To study
the effect of the ablation of the
Phe5-Gln6-Gln7-Ile8
motif on the recycling of GLUT4 to the GSC, clonal 3T3-L1 fibroblasts stably expressing GLUT4(Ser5) were developed. Their
staining with pAb828 showed that GLUT4(Ser5) was
distributed between the GSC, numerous punctuate cytoplasmic structures,
and the plasma membrane (Fig.
8A). The steady-state distribution of GLUT4(Ser5) was therefore similar to that
of GLUT4, except for its slight retention at the plasma membrane
(compare Figs. 3A and 8A; see also Fig.
1B).
In dramatic contrast with the results of studies on GLUT4 recycling,
when fibroblasts stably expressing GLUT4(Ser5) were
incubated at 37 °C for 1 h (Fig. 8B) or 18 h (data
not shown) with mAb16B12 and after their wash further incubated for 1 h in plain complete medium, the antibody was exclusively
localized to punctuate structures scattered throughout the cytoplasm.
Essentially the same results were obtained when the
GLUT4(Ser5) molecules exposed on the cell surface were
biotinylated using Bio-LC-ATB-BMPA and their distribution was studied
after internalization using FITC-streptavidin (Fig. 8C).
Furthermore, double staining of the fibroblasts for the antibody and
GLUT4(Ser5) revealed that the endocytosed antibody was
completely excluded from the perinuclear GSC (Fig. 8D). This
exclusion of the antibody was in contrast with the detection of
GLUT4(Ser5) in the GSC (Fig. 8, compare A with
B and C; see "Discussion"). Altogether the
results of these internalization studies strongly indicated that the
ablation of the
Phe5-Gln6-Gln7-Ile8
motif inhibited the targeting of endocytosed GLUT4 to the GSC.
To further characterize the vesicles that retained the antibody-tagged
GLUT4(Ser5) in the same antibody internalization experiment
described above, the fibroblasts were double-stained for the
endocytosed antibody and Rab5b (50) or LIMPIII (51), markers of early
and late endosomes/lysosomes, respectively. Their study by IMF
microscopy showed no significant overlapping between the distributions
of the mAb16B12 and Rab5b (Fig. 8E), whereas the antibody
was often found in LIMP III-positive vesicles (Fig. 8,
F-H). These results suggested that the antibody-tagged
GLUT4(Ser5) was at some point deflected from the recycling
pathway to the degradative pathway. This conclusion was in agreement
with the low levels of mAb16B12 measured in LDM extracted from
fibroblasts stably expressing GLUT4(Ser5) (Fig.
2B) and with the rapid decrease in the fluorescence
intensity of the vesicles loaded with antibody when following the
antibody internalization the fibroblasts were incubated for
1 h in plain complete medium (not shown). When the antibody
internalization experiment described above was repeated in ADL cells
stably expressing GLUT4(Ser5), again, the endocytosed
antibody was retained in vesicles scattered throughout the cytoplasm
and was excluded from the GSC (data not shown).
Expression of GLUT4 Opens the Access of pAbEL to the GSC of
Fibroblasts Stably Expressing GLUT4(Ser5)--
We next
examined the possibility that in clonal 3T3-L1 fibroblasts the failure
of the antibody-tagged GLUT4(Ser5) molecules to gain access
to the GSC was due to a dysfunction of the transport mechanisms rather
than to the ablation of the Phe5-Gln6-Gln7-Ile8
motif. For this purpose, we tested the recycling of GLUT4 in 3T3-L1
fibroblasts stably expressing GLUT4(Ser5) that were
transfected for 20 h with His/HA-tagged GLUT4 and then incubated
with pAbEL for 1 h at 37 °C. Their study by IMF microscopy
after the staining of GLUT4 and pAbEL with a mouse anti-His mAb and a
goat anti-rabbit pAb, respectively, showed that the endocytosed
antibody was divided between numerous punctuate structures scattered
throughout the cytoplasm and the GSC (Fig. 8, I and
K), whereas the bulk of the transiently expressed
His/HA-tagged GLUT4 was retained in the GSC (Fig. 8, J and
K). This different distribution demonstrated that
endocytosed GLUT4 was targeted to the GSC in the same cells that
retained the endocytosed GLUT4(Ser5) in vesicles and
therefore showed that the failure of GLUT4(Ser5) to return
to the GSC was provoked by the inactivation of the Phe5-Gln6-Gln7-Ile8 motif.
Ablation of the
Phe5-Gln6-Gln7-Ile8
Motif Provokes the Entrance of GLUT4(Ser5) into the
Degradative Pathway and Accelerates Its Turnover--
To further
investigate the accumulation of endocytosed GLUT4(Ser5) in
LIMPIII positive vesicles, the download of dextran beads into the
vesicles previously loaded with antibody-tagged GLUT4(Ser5)
was monitored and the turnovers of GLUT4 and GLUT4(Ser5)
were compared. Fibroblasts stably expressing GLUT4(Ser5)
were, for that purpose, preincubated at 37 °C for 1 h with
FITC-pAbEL before incubation for 10 min with Texas red-conjugated
dextran beads and, after washing the beads, further incubated for 50 min with plain complete medium. The distributions of the antibody and
the beads were then studied by confocal microscopy after fixation of
the cells in 4% paraformaldehyde. Their study revealed an extensive overlapping of their distributions (Fig.
9, A-D), thus indicating that
the dextran beads were downloaded into the vesicles retaining the
endocytosed GLUT4(Ser5). Because dextran beads are
transported all the way down to late endosomes/lysosomes, we concluded
that GLUT4(Ser5) was transported to these degradative
compartments. Interestingly, when the same experiment was repeated with
Tfn instead of dextran beads, the majority of the vesicles preloaded
with the antibody were not stained with Texas red-Tfn (Fig. 9,
E-H). This result, which agreed with the different
distribution of the GLUT4(Ser5)/mAb16B12 complexes and
Rab5b, strongly supported the above conclusion.
To independently asses the download of GLUT4(Ser5) into
late endosomes/lysosomes, we compared its turnover with that of GLUT4. For these purpose the two proteins were metabolically labeled in
vivo for 10 min or 1 h with
[35S]methionine/cysteine and then chased for 0, 4, 8, 12, and 24 h with an excess of cold amino acids. The study was
performed in cells incubated with and without the proteases inhibitors
chloroquine (50 mM) and leupeptin (100 µg/ml), before and
during the labeling and chase periods. The radiolabeled proteins were
immunoprecipitated with mAb16B12 and resolved by SDS-PAGE, and their
turnovers were analyzed by autoradiography. The results showed that
GLUT4(Ser5) was synthesized faster and twice more rapidly
degraded than GLUT4 (Fig. 10,
A and B) and that
the mixture of chloroquine and leupeptine slowed its degradation to a
rate comparable with that of GLUT4. Altogether, the codistribution of
endocytosed GLUT4(Ser5)/pAbEL complexes with dextran beads
and the accelerated turnover of GLUT4(Ser5) as compared
with GLUT4 indicated that after its internalization GLUT4(Ser5) was deflected to lysosomes.
Ablation of the
Phe5-Gln6-Gln7-Ile8
Motif Provokes the Redistribution of GLUT4 and Endocytosed pAbEL among
LDM Vesicles--
The localization of the bulk of GLUT4 to a
population of LDM vesicles that appeared to develop during
differentiation of fibroblasts into adipocytes and exhibited an
insulin-elicited release of GLUT4 in response to insulin (34) prompted
us to compare the distribution of GLUT4 and GLUT4(Ser5)
among the pool of LDM vesicles. For this purpose, LDM vesicles isolated
from 3T3-L1 fibroblasts stably transfected with GLUT4 or
GLUT4(Ser5) and ADL cells expressing endogenous GLUT4 were
fractionated on 12-30% sucrose velocity gradients and the
distribution profile of the proteins studied by Western analysis using
the anti-GLUT4 pAb828. It was observed that the distribution profiles
of endogenous and transfected GLUT4 through the gradients were
identical (Fig. 11, compare
A and B,
left panel) and similar to the profile displayed by the
GLUT4 compartment developed during adipocyte differentiation (34),
72 ± 3% of the transporter being accumulated between
fractions 6 and 11. These results strongly suggested that the GSC
already existed in 3T3-L1 fibroblasts and agreed with results pointing the universality of the GLUT4 distribution (20, 41, 51) (see
"Discussion"). Interestingly the Rab isoform, Rab4, often associated with the translocation of GLUT4 to the plasma membrane (53-56), was excluded from the fractions containing the majority of
intracellular GLUT4, which instead contained significant amounts of
Rab5b, the Rab isoform involved in endocytosis (Fig. 11C).
When the same study was repeated in 3T3-L1 fibroblasts stably
expressing GLUT4(Ser5), it was observed that, in contrast
to GLUT4, the mutant was more uniformly distributed throughout the
gradient, with only 49 ± 2.8 of it being accumulated between
fractions 6 and 11 (Fig. 11D, left panel).
To further investigate the effect of the Ser5 mutation on
the distribution of GLUT4 among LDM vesicles, we compared the gradient profiles of endocytosed pAbEL, GLUT4, and GLUT4(Ser5) in
fibroblasts stably expressing the transfected proteins. Except for some
antibody recovered with the top fractions, the gradient profiles of
GLUT4 and the antibody essentially overlapped (Fig. 11B,
compare left and right panels). In contrast, the
distributions of GLUT4(Ser5) and the antibody were sharply
different, and the majority of pAbEL was recovered with the lighter
fractions (Fig. 11D, compare right and left
panels). To study whether the difference between the distributions
of GLUT4(Ser5) and the antibody were the result of their
dissociation, we measured the fraction of pAbEL that remained bound to
the GLUT4(Ser5) recovered with LDM vesicles. For this
purpose, LDM vesicles loaded with pAbEL and extracted with 1% Triton
X-100 were incubated with anti-GLUT4 pAb828 covalently bound to
Sepharose, and the levels of antibody were measured in the supernatant
and pellet separated by low speed centrifugation. Their study showed
that the bulk of endocytosed antibody recovered with LDM vesicles
remained bound to GLUT4(Ser5) (Fig. 11E,
left panel). The same result was observed when the stability
of the antibody/GLUT4 complex was investigated (Fig. 11E,
right panel). Because the distribution of the antibody
appeared to reflect the distribution of endocytosed
GLUT4(Ser5) among LDM vesicles, we concluded that
endocytosed GLUT4(Ser5) moved through a small pool of light
LDM vesicles.
To identify the structural motifs involved in the recycling of
GLUT4 from the cell surface to the GSC as well as to characterize this
compartment, we have compared the cellular distributions of
antibody/biotin tagged GLUT4 with active and ablated
Phe5-Gln6-Gln7-Ile8
motif. It is important to note that the trafficking of GLUT4 was not
disturbed by the tags used because (a) the results obtained with two different antibodies and biotin were identical and
(b) the patterns of distribution of GLUT4 and
GLUT4(Ser5) carrying the same tags were different.
Our studies show a dramatic difference between the distributions of
endocytosed GLUT4 and GLUT4(Ser5). According to the
results, the bulk of endocytosed tagged GLUT4 complexes is downloaded
in a reticular compartment localized to one pole of the nucleus and
juxtaposed to the Golgi complex, as shown by double-immunostaining of
cells with antibodies against mAb16B12 and the Golgi marker MG160,
whereas tagged GLUT4(Ser5) is targeted to the lysosomal
degradative pathway.
With regard to the morphology, localization, and links of the GSC with
the Golgi complex, the GSC can be described as an organelle constituted
by flat saccular structures with multiple fenestration in their walls
and arranged as a reticulum at one of the nuclear poles, all of them
characteristics often associated with the Golgi cisternae (43) The
previous localization of the bulk of intracellular GLUT4 in cisternae
and tubulovesicular structures clustered on the trans side
Golgi is consistent with these observations (7, 8, 57). The
localization of endocytosed mAb16B12/GLUT4 complexes in a structure
separated from GMPt In agreement with a recent study we have observed that the bulk of
intracellular GLUT4 is retained within a pool of LDM vesicles with a
Among the organelles jamming the perinuclear area can be counted those
constituting the secretory pathway, the structures involved in the
recycling of endocytosed and transcytosed proteins, late endosomes, and
lysosomes. It was therefore important to examine whether the GSC has
morphological, physical, and functional properties distinct from those
neighboring compartments. As discussed above, the immunostaining of
fibroblasts and ADL cells with specific antibodies against GLUT4 and
the Golgi marker MG160 clearly shows that the two organelles, although
closely juxtaposed, are distinct. Furthermore, the codistribution of
endocytosed GLUT4 and Tfn in vesicles and the decrease in the number of
these following the chase of two endocytosed molecules agree with
previous observations describing their endocytosis and transport to the
perinuclear area packed in the same vesicles (40-42). On the other
hand, the sealing of the GSC to Tfn disagrees with the results of
previous studies performed by conventional IMF microscopy, which
described as identical the perinuclear distributions of GLUT4 and the
Tfn receptor (41) and tends to agree with the segregation of GLUT4 and
the Tfn receptor in skeletal myotubes (57). The emergence of the GSC as
an extended reticular structure among the pleiade of vesicles loaded
with Tfn and GLUT4 dramatically illustrates the distinct nature of the
GSC and the recycling compartment traversed by Tfn. Furthermore, the
different distributions of GLUT4 and GLUT4(Ser5) and the
identification of the vesicles that retain GLUT4(Ser5) as
late endosomes/lysosomes show that GSC and lysosomes are distinct organelles.
The detection of GLUT4(Ser5) in the GSC and the exclusion
of the internalized antibody/GLUT4(Ser5) complexes from
that opens the question of what is the source of the
GLUT4(Ser5) molecules detected in the GSC. There could be
at least two not mutually exclusive sources: newly synthesized
molecules and molecules recycled through a pathway that excludes the
plasma membrane. With regard to the first possibility, we have noted in
3T3-L1 fibroblasts stably expressing GLUT4 that were loaded for 1 h with FITC-conjugated anti-GLUT4 pAbEL and then chased for 1 h
with antibody-free medium that further incubation for 1, 2, and 3 h with anti-FITC antibodies did not decrease significantly the staining of the GSC. This observation, together with the relative rapid recycling of endocytosed GLUT4 to the GSC, strongly suggest that in
unstimulated cells the targeting of GLUT4 from the GSC to the plasma
membrane is slow as compared with its recycling. Although the retention
of internalized GLUT4(Ser5) in endosomes, as precluded to
compare the speed of these two processes in cells transfected with of
GLUT4(Ser5), a difference comparable with that found in the
GLUT4 studies could explain the detection of GLUT4(Ser5) in
the GSC because this would never completely depleted of
GLUT4(Ser5) molecules. With respect to the possibility that
a pool of GLUT4(Ser5) and, perhaps, also of GLUT4 molecules
could be recycled to the GSC through a pathway that excludes the
plasma, although it is an attractive possibility, it requires a
technology that, as far as we know, is not available.
While this study was in progress Melvin et al. (24) reported
that the inactivation of the
Phe5-Gln6-Gln7-Ile8 in
adipocytes results in retention of GLUT4 in an endosomal compartment that can be partly ablated in cells incubated for 3 h with Tfn-HRP before developing the peroxidase reaction. This and our observations suggest that the
Phe5-Gln6-Gln7-Ile8
motif mediates a transport step in endosomes. The different
distributions of endocytosed mAb16B12/GLUT4(Ser5) complexes
and Tfn, the down load of dextran beads into the vesicles that retain
GLUT4(Ser5), and the staining of these vesicles with an
anti-LIMPIII antibody, indicate, however, that the mutant is
transported to the late endosomes/lysosomes. This conclusion is also
supported by the very rapid degradation of GLUT4(Ser5) as
compared with GLUT4 and the slowing of its degradation by drugs
inhibitory of lysosomal proteases.
Altogether the studies on the GLUT4(Ser5) distribution
strongly suggest that the transport step mediated by the
Phe5-Gln6-Gln7-Ile8
motif probably lays at or near the cross-road of the recycling and
degradative pathways. Sorting out of GLUT4(Ser5) of the
recycling pathway may occur as a result of its retention in the sorting
endosome as it matures into a late endosome. This could explain its
lysosomal fate and degradation in these. Further work is required to
characterize the organelle and the machinery involved in the sorting of
GLUT4 at this cross-road. The intracellular reading of the
Phe5-Gln6-Gln7-Ile8
motif in an endocytic compartment is novel because the transport steps
associated with F(Y)XXZ activity has been mainly localized to the TGN and the plasma membrane.
Two features of the steady-state distribution of
GLUT4(Ser5) deserve further comment, the detection of
GLUT4(Ser5) at the GSC and its low but significant
accumulation at the plasma membrane. Although it is likely that a 2- or
3-fold increase in the turnover of GLUT4(Ser5) may not
sufficiently decrease the staining of the GSC with anti-GLUT4 antibodies as to be detected by microscopy, we have observed that the
levels of GLUT4(Ser5) in the pool of LDM vesicles that
retain GLUT4 are significantly decreased.
The faint staining of the plasma membrane of fibroblasts stably
expressing GLUT4(Ser5) immunostained with anti-GLUT4
antibodies as well as the partial digestion of GLUT4(Ser5)
by the trypsin added to the incubation medium, indicate that GLUT4(Ser5) is significantly retained at the plasma
membrane. Previous studies have involved the NH2 tail of
GLUT4 and, specifically, the
Phe5-Gln6-Gln7-Ile8
motif (16, 19), in the internalization of GLUT4. It should be pointed,
however, that the GLUT4(Ala489-Ser490) and
GLUT4(Ser5-Ala489-Ser490) mutants
accumulated in much larger amounts onto the cell surface as compared
with GLU4(Ser5) and that both failed to internalize
significantly mAb16B12 and pAbEL,2 therefore suggesting
that GLUT4 endocytosis is mediated by the carboxy
Leu489-Leu490-based motif. In view of the new
data suggesting that the
Phe5-Gln6-Gln7-Ile8
motif mediates a sorting step localized to endosomes, it will be
interesting to study whether the slight accumulation of
GLUT4(Ser5) onto the cell surface is produced by the
partial recycling to the cell surface of the molecules accumulated in endosomes.
The different cellular distributions of GLUT4 and
GLUT4(Ser5) and the possibility to load the organelles that
retain these two molecules with probes (i.e. HRP-conjugated
antibodies) successfully used in organelle isolation (52) should help
progress in their biochemical characterization. As part of this effort,
it will be interesting to compare the biochemical properties of the GSC isolated from cells expressing or not the transporter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 µg/ml insulin, 10 µg/ml biotin, 0.25 µM dexamethasone, and 500 µM 1-isobutyl
3-methylxanthine) and then with 10 -5 M
insulin in complete medium for different time periods as required. The
fibroblasts so treated developed large lipid-droplets in their cytoplasm and will be referred henceforth as adipocyte-like (ADL) cells. ADL cells were fixed with 4% paraformaldehyde and stained with
1 mM Nile blue prepared in phosphate-buffered saline to
monitor the development of lipid droplets in the cytoplasm. ADL cells fixed with paraformaldehyde were permeabilized for 3 min with cold
(
20 °C) methanol and immunostained with specific antibodies as
required for their study by immunofluorescence (IMF) microscopy.
1 was developed
in the laboratory, and its properties have been reported (27)
Fluorescein and Texas-red conjugated antibodies were from Cappel
(Durham, NC). HRP-conjugated goat anti-mouse and F(ab')2
donkey anti-rabbit antibodies were from Amersham Pharmacia Biotech.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, HA-tagged GLUT4 and
GLUT4(Ser5). GLUT4(Ser5) was developed by
site-directed mutagenesis. The mutated residue, named by the one-letter
code, and the HA epitope (YPYDVPDYADD), inserted into the exofacial
loop (black box), are shown in position in a diagram of
GLUT4. B, time course of endogenous GLUT4 expression during
in vitro differentiation of 3T3-L1 fibroblasts into ADL
cells. Untransfected 3T3-L1 fibroblasts were incubated for 7 days in
IDBX medium and then for the indicated times in complete medium
containing 10 5 insulin. The cellular levels of GLUT4 were
monitored by Western blot analysis using pAb828 and the ECL technique.
C and D, surface levels of transfected GLUT4 and
GLUT4(Ser5). 3T3-L1 fibroblasts (C) and ADL
cells (D) either untransfected or stably transfected with
GLUT4 or GLUT4(Ser5) were grown to 90% confluence for
72 h in complete medium on 100-mm dishes. ADL cells were further
incubated for 4 h in DMEM and then for 40 min in DMEM with or
without 10
7 M insulin. The cells were
poisoned for 20 min at 37 °C with 2 mM KCN before their
incubation for 10 min at 37 °C with 2.5 mg/ml trypsin, and membranes
were prepared as described under "Experimental Procedures." The
relative surface levels of the transfected proteins in 30 µg of
membrane protein were quantitated by scanning of Western blots
developed using pAb828 and the ECL technique. Shown in the figure is
one of three separate experiments. Mean values were calculated from the
three experiments. Percentages of GLUT4 and GLUT4(Ser5) in
the surface of 3T3-L1 fibroblasts were 5.5 ± 1 and 22 ± 2.1%, respectively, whereas in ADL cells incubated without insulin
they were 6.5 ± 0.5 and 38 ± 2%, and after incubation with
insulin they were 71 ± 2 and 53 ± 3%. wt, wild
type.
7 M insulin, the levels of GLUT4 in the
plasma membrane went up, as shown by the increase in its digestion by
trypsin, nearly 10-fold (Fig. 1D). Interestingly, the
increase in the surface levels of GLUT4(Ser5) in response
to insulin, less than 2-fold, was significantly smaller than the
increase in GLUT4 (Fig. 1D).
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Fig. 2.
Wild-type GLUT4 and
GLUT4(Ser5)/mAb16B12 complexes remain stable at low
pH. Clonal 3T3-L1 fibroblasts stably expressing GLUT4
(A, upper panel) and GLUT4(Ser5)
(A, lower panel) and labeled for 1 h with
[35S]methionine/cysteine were chased for 1 h with
complete medium and solubilized membrane proteins prepared as described
under "Experimental Procedures." Immunoprecipitates developed by
incubation of whole solubilized membranes with 5 µl of
mAb16B12/protein G-Sepharose were incubated for 10 min or 1 h with
50 mM Tris-HCl, pH 7.4, or with 50 mM
cacodylate buffer, pH 6.4 or 5.5, containing 100 mM NaCl
and 2 mM EDTA. After centrifugation for 1 min at
14,000 × g, the proteins in the supernatants
(s) and pellets (p) were resolved by SDS-PAGE,
and their content in GLUT4 and GLUT4(Ser5) was analyzed by
autoradiography. In a similar experiment performed with pAbEL, it was
observed that GLUT4 and GLUT4(Ser5)/pAbEL complexes
remained stable at pH 7.4, 6.4 and and 5.5 (data not shown).
Endocytosed mAb16B12 accumulates in LDM vesicles in a manner mediated
by the activity of the
Phe5-Gln6-Gln7-Ile8
motif. 3T3-L1 fibroblasts untransfected or stably expressing GLUT4,
GLUT4(Ser5) or were incubated for 1 h at 37 °C with
mAb16B12 prepared to 50 µg/ml in complete medium, and the levels of
antibody in the LDM and HDM fractions were studied by Western blot
analysis using a HRP goat anti-mouse pAb and the ECL technique
(B). Comparable results were obtained in experiments
performed with pAbEL.
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Fig. 3.
Endocytosed mAb16B12 accumulates in the
perinuclear GSC of 3T3-L1 fibroblasts stably expressing GLUT4.
Clonal 3T3-L1 fibroblasts stably expressing HA-tagged GLUT4
(A) were either preincubated for 1 h at 37 °C with
50 µg/ml mAb 16B12 (B and D-F) or surface
biotinylated using Bio-LC-ATB-BMPA (C). After their wash and
their incubation for 1 h at 37 °C in plain complete medium, the
cells were either fixed or incubated for the indicated times in 10 mg/ml BFA (F-I). The distributions of GLUT4 (A,
C, E, and G), endocytosed mAb16B12
(B and D-F), and the Golgi marker MG160
(D, H, and I) were studied by IMF
microscopy. Cells were single (A-C, H, and
I) or double-stained (D-G). GLUT4 was studied
with pAb828 (A and G), anti-HA pAb Y11
(E), or FITC-conjugated streptavidin (C). Second
antibodies were goat anti-mouse or anti-rabbit antibodies conjugated to
FITC, Texas red, or horseradish peroxidase (B,
inset). Images in D and E were
simultaneously photographed through fluorescein and Texas red and
processed using the Image-Pro 2.0 program. The tubular extensions of
the GSC and Golgi produced in response to BFA are marked with
white arrows. n, nucleus. The
bars indicate: 12 µm (A), 11.4 µm
(B), 7.8 µm (C), 0.8 µm (D and
E), 3 µm (F-H), and 4.5 µm
(I).
1 (27) were
compared by confocal microscopy; the two proteins showed very little
overlapping, thus indicating that they were localized in different
cellular compartments (Fig. 4). To
further characterize the compartment that retained the antibody, cells
incubated for 1 h with mAb16B12 were double-stained for the
endocytosed antibody and for those GLUT4 molecules that retained in the
GSC were not exposed onto the cell surface. For this purpose
endocytosed mAb16B12/GLUT4 complexes were cross-linked with 4%
paraformaldehyde and stained wit FITC goat anti-mouse pAb, whereas the
GLUT4 molecules retained in the GSC and carrying antibody-free HA tags
were stained with the anti-HA pAbY11 and a Texas red goat anti-rabbit
pAb. The colocalization of mAb16B12 and pAbY11 to the same perinuclear
reticular structure (Fig. 3E) led us to conclude that the
endocytosed mAb16B12/GLUT4 complexes were targeted to the GSC.
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Fig. 4.
Endocytosed mAb16B12 accumulates in a
perinuclear compartment separate from the TGN. Clonal 3T3-L1
fibroblasts stably expressing HA-tagged GLUT4 were preincubated for
1 h at 37 °C with 50 µg/ml mAb16B12 and after their wash and
their incubation for 1 h at 37 °C in plain complete medium
fixed, double stained for mAb16B12, with an FITC-conjugated goat
anti-mouse antibody, and for the TGN protein membrane marker
GMPt 1, with a Texas red goat anti-rabbit antibody, and
their distribution was studied by confocal microscopy. The cell marked
in C-F with an arrow is shown in A
and B at larger magnification. The cell marked with an
asterisk was not transfected with HA-tagged GLUT4.
Digits in the lower left corner of each panel
indicate the distance (µm) of the sections to the cell bottom.
Bars, 9.7 µm.
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Fig. 5.
The GSC is distinct from the recycling
compartment traversed by endocytosed transferrin. Clonal 3T3-L1
fibroblasts stably transfected with HA-tagged GLUT4 and preincubated
for 1 h with FITC-pAbEL were washed and incubated for 10 min with
Texas red-Tfn (50 µg/ml) before incubation for 10 (A-E)
or 50 min (F) with plain complete medium. After
fixation with 4% paraformaldehyde, the cells were studied by
fluorescence confocal microscopy. A-D are
enlargements of the perinuclear area from sections of the cell
shown in C at low magnification. Note the exclusion of Tfn
from the the reticular GSC loaded with pAbEL (A-D).
Digits in the lower left corner of each panel
indicate the distance (µm) of the sections to the cell bottom.
Bars, 12 µm.
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Fig. 6.
GSC ultrastructure. 3T3-L1 fibroblasts
stably transfected with HA-tagged GLUT4 grown for 2 days to 90%
confluence on 60 mm plastic dishes were incubated for 1 h at
37 °C with pAbEL diluted to 50 µg IgG/ml in complete medium. After
inhibition of endogenous peroxidase the cells were stained with a
HRP-conjugated F(ab')2 donkey anti-rabbit antibody, using
the pre-embedding method. Boxed areas in A,
C, and E are shown at large magnification in
B, D, and F. Saccular structures with
characteristic fenestration are shown in F and G.
The bars indicate: 0.11 µm (A, C,
and E) and 0.16 µm (B, D, and
F-H).
5 M insulin that were preincubated for
2 h in DMEM and then treated for 40 min with 10
7
M insulin. The staining of these cells for GLUT4 showed
that the majority of the endogenous protein was retained in a structure organized on one of the nuclear poles (Fig.
7A). Furthermore, as described
for transfected GLUT4 in fibroblasts (Fig. 3) (20), the structure
retaining GLUT4 colocalized with the Golgi stained with the anti-MG160
antibody (Fig. 7, compare A and B). In addition, GLUT4 was also detected in the plasma membrane (Fig. 7A,
arrows). Moreover, as in fibroblasts, treatment of ADL cells
for 1 h with 10 µg/ml BFA provoked the tubulation but did not
cause the disappearance of the GSC (Fig. 7C).
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Fig. 7.
Surface internalized mAb16B12 accumulates in
the GSC of ADL cells. 3T3-L1 adipocytes expressing endogenous
GLUT4 preincubated for 2 h in DMEM and then incubated for 40 min
with 10 7 M insulin were double-stained for
endogenous GLUT4 (A) and the Golgi marker MG 160 (B). In a separate experiment 3T3-L1 adipocytes were
incubated for 1 h with 10 µg/ml BFA and stained for GLUT4
(C). The stained plasma membrane (A) and the
tubular extensions of the GSC (C) are marked with
arrows. 3T3-L1 fibroblasts stably expressing HA-epitope
tagged GLUT4 were differentiated into ADL cells as described under
"Experimental Procedures." At days 3 (D) and 6 (E-G) of incubation in IDBX medium, the fibroblasts were
stained for lipids using Nile blue. ADL cells incubated for 6 days in
IDBX medium were loaded with mAb16B12 as described in the legend to
Fig. 3, and the distributions of endocytosed antibody (F)
and GLUT4 (G) were studied by IMF microscopy as described in
the legend to Fig. 3. The cell marked with an arrow in
F is shown in the inset enlarged and as black and
white to facilitate the observation of the large cytoplasmic lipid
droplets. The bars indicate: 6.5 µm (A and
C), 7 µm (D and E), and 8 µm
(F).
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Fig. 8.
Ablation of the
Phe5-Gln6-Gln7-Ile8
motif impedes access of internalized mAb16B12 to the GSC. Clonal
3T3-L1 fibroblasts stably expressing GLUT4(Ser5) were
incubated in normal medium (A and C) or with
mAb16B12 (B and D-H) as described in the legend
to Fig. 3. The steady-state distribution of GLUT4(Ser5) was
studied using pAb828 and a FITC-goat anti-rabbit pAb (A).
The distributions of mAb16B12 and Bio-LC-ATB-BMPA tagged GLUT4 were
studied with FITC-goat anti-mouse pAb (B, D,
E, G, and H) and FITC-streptavidin
(C), respectively. Cells were single (A-C) or
double stained for mAb16B12 and GLUT4 (D), Rab5b
(E), or LIMPIII (F and H) using
specific pAbs and a goat anti-rabbit pAb conjugated to Texas-red.
Boxed areas in F-H are shown enlarged in the
inserts in the same panels. Expression of GLUT4 opens the access of
pAbEL to the GSC in fibroblasts stably expressing
GLUT4(Ser5). Clonal 3T3-L1 fibroblasts stably expressing
GLUT4(Ser5) were transiently transfected for 20 h with
HIS-tagged GLUT4 and after their incubation for 1 h with pAbEL
(prepared in complete medium to ~50 µg IgG/ml) processed and
double-stained for the antibody, using a FITC-goat anti-rabbit pAb
(I and K) and for His-GLUT4, using a mouse
anti-His mAb and a Texas red-goat anti-mouse pAb (J and
K). The cells were separately (I and
J) or simultaneously (K) photographed through
fluorescence. The bars indicate: 12 µm (A), 5 µm (B and E), 3 µm (C and
D), 4 µm (F-H), and 4.8 µm
(I-K).
View larger version (43K):
[in a new window]
Fig. 9.
Endocytosed dextran is downloaded into the
compartment retaining GLUT4(Ser5). Cells stably
transfected with GLUT4(Ser5) were preincubated with
FITC-pAbEL for 1 h before incubation for 10 min with Texas
red-dextran beads (molecular weight 70,000; at 0.1 mg/ml) and after
their wash further incubated for 50 min in plain complete medium
(A-D). The same experiment was repeated substituting Texas
red-Tfn for dextran beads (E-H). Note the overlapping
between the endocytosed antibody and the dextran beads distributions
and the segregation of the antibody and Tfn into different punctuate
organelles. Digits in the lower left corner of
each panel indicate the distance (µm) of the sections to the cell
bottom. Bars, 9.7 µm.
View larger version (18K):
[in a new window]
Fig. 10.
Inactivation of the
Phe5-Gln6-Gln7-Ile8
Motif Increases the Turnover of GLUT4. 3T3-L1 fibroblasts stably
expressing GLUT4 ( ) or GLUT4(Ser5) (
,
) and grown
to 80% confluence were metabolically labeled for 1 h
(A and B) or for 10 min (C) with
[35S]methionine/cysteine (>1,000 Ci/mmol) and chased for
0, 4, 8, 12, and 18 h (A). To study the effect of the
inhibition of lysosomal proteases on the GLUT4(Ser5)
turnover, 50 mM chloroquine and 100 µg/ml leupeptin were
included during the preincubation, labeling, and chase periods (
).
Radiolabeled GLUT4 and GLUT4(Ser5) were immunoprecipitated
with mAb16B12 and resolved by SDS-PAGE. The labeling intensity of the
precursor (p) and mature (m) GLUT4 and
GLUT4(Ser5) species was quantitated by scanning, and the
resulting values were plotted against the chase periods (B).
In A, the labeling of the precursor species (0 min chase) is
shown at two different exposures. The experiments shown are
representative of three separate experiments.
View larger version (41K):
[in a new window]
Fig. 11.
Ablation of the
Phe5-Gln6-Gln7-Ile8
motif provokes the redistribution of GLUT4 and endocytosed pAbEL among
LDM vesicles. 3T3-L1 adipocytes expressing endogenous GLUT4 and
clonal 3T3-L1 fibroblasts stably expressing GLUT4 or
GLUT4(Ser5) were grown for 72 h to 90% confluence on
100-mm plastic dishes and then incubated with 3 ml of pAbEL prepared to
50 µg IgG/ml in complete medium for 1 h at 37 °C. LDM loaded
with pAbEL were prepared as described (33) and fractionated by
centrifugation on 12-30% sucrose gradients (see "Experimental
Procedures"). The gradient profiles of GLUT4 (A and
B, left panel), Rab4 and Rab5b (C),
GLUT4(Ser5) (D, left panel), and
endocytosed pAbEL (B and D, right
panels) were studied by Western analysis. GLUT4 and
GLUT4(Ser5) were probed with pAb828 and a second HRP goat
anti-rabbit antibody using the ECL technique. Endocytosed pAbEL was
directly probed with a HRP-conjugated goat anti-rabbit antibody,
whereas the endosomal markers Rab4 and Rab5b were probed with specific
rabbit pAb and a HRP-conjugated goat anti-rabbit antibody. The
percentages of GLUT4 (72 ± 3%) and GLUT4(Ser5)
(49 ± 2.8%) accumulated in fractions 6-11 were calculated from
values measured by scanning the ECL developed films from three separate
experiments. LDM enriched fractions loaded with pAbEL prepared from
3T3-L1 fibroblasts stably expressing GLUT4(Ser5) and GLUT4
were solubilized in buffer B and then incubated overnight at 4 °C
with Sepharose alone ( ) or with anti-GLUT4 pAb828 covalently bound to
Sepharose (+) in buffer B containing 0.5% Triton X-100, and the
fractions of free (s) and bound pAbEL (p) were
separated by low speed centrifugation and analyzed by Western blot
using a HRP-conjugated goat anti-rabbit antibody (E).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, a TGN membrane protein that resides
and stains uniformly all the TGN subcompartments (27), strongly
suggests that the GSC is a compartment distinct from the TGN. It is,
however, very little of what we know regarding the nature,
organization, and boundaries of the TGN, an organelle with a very
complex organization defined as the site of sorting of the proteins
that traverse the Golgi. To define the boundaries between the TGN and
adjacent compartments requires the use of antibody markers that are not
always available. An important difference, however, between the Golgi
and the neighboring compartments is their distinct sensitivity to BFA.
The relatively rapid disassembly of all the Golgi parts in response to
BFA and the resistance of the GSC to the drug, also noted on skeletal
myotubes (57) and a characteristic of the vacuolar compartments
localized beyond the Golgi involved in the transport of proteins to the
cell surface (36-38), strongly suggest that the GSC is a compartment
separated from the Golgi. This conclusion is also supported by the
recent demonstration that GLUT4 is targeted from the TGN to the GSC by a mechanism involving the carboxy
Leu489Leu490-based motif (20).
0 = 1.072-1.083 (34). The recovery of the endocytosed antibody with this pool of vesicles points to these as the most likely
constituents of the GSC. Furthermore, our studies on 3T3-L1 fibroblasts
stably transfected with GLUT4 and adipocytes expressing endogenous
GLUT4, indicate that the profiles of LDM fractionated by differential
centrifugation on sucrose gradients are comparable (Fig. 11, compare
A and B, left panel). This result
strongly suggests that the GSC already exists in 3T3-L1 fibroblasts, a
result also confirmed by the IMF microscopy studies. The universality
of the GSC, repeatedly demonstrated by studies that examined the
distribution of transfected GLUT4 in a variety of cells (20, 21, 41, 58) is, however, not totally accepted. The recent demonstration that
GLUT4 is expressed in specific sets of neurons (44-49) and within them
in structures with the morphology characteristic of Golgi saccules (44)
shows that GLUT4 is not exclusively expressed in insulin-responsive
cells and tends to support the universality of the GSC distribution. We
would like to speculate that cells that do not express GLUT4 in
vivo use the GSC to regulate the store and trafficking of other
protein loads. Cells expressing GLUT4 in vivo might contain
in their GSC a specific set of proteins used to respond to changes in
glucose homeostasis by controlling the access, retention, and exit of
GLUT4. This set of proteins could be replaced in cells that do not
express GLUT4 by other sets designed to regulate the store and
trafficking of different protein loads. The notion of GLUT4 being
stored within a single compartment is not new, but a model in which the
transporter is stored in scores of vesicles ready to fuse with the
plasma membrane has also met ample support. The recent demonstration
that the rate-limiting step for insulin-stimulated GLUT4 translocation is the trafficking of GLUT4 vesicles and not their fusion with the
plasma membrane (59) appears to fit more with a model in which GLUT4 is
released from a store compartment, packed in vesicles, and transported
to the cell surface.
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FOOTNOTES |
---|
* This work was supported by Ministerio Español de Educación y Ciencia Grant PB94-0035 and by European Commission Grants ERB4061PL95-0924 and FMRX-CT96-0058.
Supported by a fellowship from the Ministerio of Ciencia y
Tecnología and European Program Grant ICA4-CT-1999-10001.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.
§ Supported by European Program Grants ERB4061PL95-0924.
¶ To whom correspondence should be addressed: Centro de Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain. Tel.: 91-3978455; Fax: 91-3974799; E-mail: isandoval@cbm.uam.es.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006739200
2 S. Palacios, V. Lalioti, and I. V. Sandoval, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: GSC, GLUT4 store compartment; HA, hemagglutinin; BFA, brefeldin A; DMEM, Dulbecco's modified Eagle's medium; ADL, adipocyte-like; IMF, immunofluorescence; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; pAb, polyclonal antibody; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; Tfn, transferrin; HDM, high density microsomes; LDM, low density microsomes; TGN, trans-Golgi network; Bio-LC-ATB-BMPA, 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1-3-bis (d-mannose4cycloxy)-2pnopylamine.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Klip, A.,
Tsakiridis, T.,
Marette, A.,
and Ortiz, P. A.
(1994)
FASEB J.
8,
43-53 |
2. | Olson, A. L., and Pessin, J. E. (1996) Annu. Rev. Nutr. 16, 235-256[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kahn, B. B. (1998) Cell 92, 593-606[Medline] [Order article via Infotrieve] |
4. | James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185[CrossRef][Medline] [Order article via Infotrieve] |
5. | James, D. E., Strube, M., and Mueckler, M. (1989) Nature 338, 83-87[CrossRef][Medline] [Order article via Infotrieve] |
6. | Schmid, S. L., and Smythe, E. (1991) J. Cell Biol. 114, 869-880[Abstract] |
7. | Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., and Lienhard, G. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7815-7819[Abstract] |
8. |
Rodnick, K. J.,
Slot, J. W.,
Studelska, D. R.,
Hanpeter, D. E.,
Robinson, L. J.,
Geuze, H. J.,
and James, D. E.
(1992)
J. Biol. Chem.
267,
6278-6285 |
9. |
Cushman, S. W.,
and Wardzala, L. J.
(1980)
J. Biol. Chem.
255,
4758-4762 |
10. |
Yang, J.,
and Holman, G. D.
(1993)
J. Biol. Chem.
268,
4600-4603 |
11. |
Jhun, B. H.,
Rampal, A. L.,
Liu, H.,
Lachaal, M.,
and Jung, C. Y.
(1992)
J. Biol. Chem.
267,
17710-17715 |
12. |
Satoh, S.,
Nishimura, H.,
Clark, A. E.,
Kozka, I. J.,
Vannucci, S. J.,
Simpson, I. A.,
Quon, M. J.,
Cushman, S. W.,
and Holman, G. D.
(1993)
J. Biol. Chem.
268,
17820-17829 |
13. | Piper, R. C., Tai, C., Kulesza, P., Pang, S., Warnock, D., Baenziger, J., Slot, J. W., Geuze, H. J., Puri, C., and James, D. E. (1993) J. Cell Biol. 121, 1221-1232[Abstract] |
14. |
Verhey, K. J.,
and Birnbaum, M. J.
(1994)
J. Biol. Chem.
269,
2353-2356 |
15. | Corvera, S., Chawla, A., Chakrabarti, R., Joly, M., Buxton, J., and Czech, M. P. (1994) J. Cell Biol. 126, 979-989[Abstract] |
16. | Garippa, R. J., Judge, T. W., James, D. E., and McGraw, T. E. (1994) J. Cell Biol. 124, 705-715[Abstract] |
17. |
Garippa, R. J.,
Johnson, A.,
Park, J.,
Petrush, R. L.,
and McGraw, T. E.
(1996)
J. Biol. Chem.
271,
20660-20668 |
18. | Yeh, J. I., Verhey, K. J., and Birnbaum, M. J. (1995) Biochemistry 34, 15523-15531[Medline] [Order article via Infotrieve] |
19. | Araki, S., Yang, J., Hashiramoto, M., Tamori, Y., Kasuga, M., and Holman, G. D. (1996) Biochem. J. 315, 153-159[Medline] [Order article via Infotrieve] |
20. |
Martinez-Arca, S.,
Lalioti, V.,
and Sandoval, I. V.
(2000)
J. Cell Sci.
113,
1705-1715 |
21. | Haney, P. M., Levy, M. A., Strube, M. S., and Mueckler, M. (1995) J. Cell Biol. 129, 641-658[Abstract] |
22. | Marsh, B. J., Alm, R. A., McIntosh, S. R., and James, D. E. (1995) J. Cell Biol. 130, 1081-1091[Abstract] |
23. | Verhey, K. J., Yeh, J. I., and Birnbaum, M. J. (1995) J. Cell Biol. 130, 1071-1079[Abstract] |
24. | Melvin, D. R., Marsh, B. J., Walmsley, A. R., James, D. E., and Gould, G. W. (1999) Biochemistry. 38, 1456-1462[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Barriocanal, J. G.,
Bonifacino, J. S.,
Yuan, L.,
and Sandoval, I. V.
(1986)
J. Biol. Chem.
261,
16755-16763 |
26. |
Gonatas, J. O.,
Mezitis, S. G.,
Stieber, A.,
Fleischer, B.,
and Gonatas, N. K.
(1989)
J. Biol. Chem.
264,
646-653 |
27. | Yuan, L., Barriocanal, J. G., Bonifacino, J. S., and Sandoval, I. V. (1987) J. Cell Biol. 105, 215-227[Abstract] |
28. |
Czech, M. P.,
and Buxton, J. M.
(1993)
J. Biol. Chem.
268,
9187-9190 |
29. |
Kunkel, T. A.
(1985)
J. Biol. Chem.
260,
12866-12874 |
30. | Reeves, R., Gorman, C. M., and Howard, B. (1985) Nucleic Acids Res. 13, 3599-3615[Abstract] |
31. | Gorman, C. M., Rigby, P. W., and Lane, D. P. (1985) Cell 42, 519-526[Medline] [Order article via Infotrieve] |
32. | Koumanov, F., Yang, J., Jones, A. E., Hatanaka, Y., and Holman, G. D. (1998) Biochem. J. 330, 1209-1215[Medline] [Order article via Infotrieve] |
33. |
Stephens, J. M.,
Lee, J.,
and Pilch, P. F.
(1997)
J. Biol. Chem.
272,
971-976 |
34. |
El-Jack, A. K.,
Kandror, K. V.,
and Pilch, P. F.
(1999)
Mol. Biol. Cell
10,
1581-1594 |
35. | Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269[Abstract] |
36. | Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S., and Klausner, R. D. (1989) Cell 56, 801-813[Medline] [Order article via Infotrieve] |
37. | Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L., and Klausner, R. D. (1991) Cell 167, 601-616 |
38. | Alcalde, J., Bonay, P., Roa, A., Vilaro, S., and Sandoval, I. V. (1992) J. Cell Biol. 116, 69-83[Abstract] |
39. | Yamashiro, D. J., Tycko, B., Fluss, S. R., and Maxfield, F. R. (1984) Cell 37, 789-800[Medline] [Order article via Infotrieve] |
40. |
Zorzano, A.,
Wilkinson, W.,
Kotliar, N.,
Thoidis, G.,
Wadzinkski, B. E.,
Ruoho, A. E.,
and Pilch, P. F.
(1989)
J. Biol. Chem.
264,
12358-12363 |
41. | Hudson, A. W., Fingar, D. C., Seidner, G. A., Griffiths, G., Burke, B., and Birnbaum, M. J. (1993) J. Cell Biol. 122, 579-588[Abstract] |
42. | Livingstone, C., James, D. E., Rice, J. E., Hanpeter, D., and Gould, G. W. (1996) 315, 487-495 |
43. | Rambourg, A., and Clermont, Y. (1986) Am. J. Anat. 176, 393-409[Medline] [Order article via Infotrieve] |
44. | El Messari, S., Leloup, C., Quignon, M., Brisorgueil, M. J., Penicaud, L., and Arluison, M. (1998) J. Comp. Neurol. 399, 492-512[CrossRef][Medline] [Order article via Infotrieve] |
45. | Kobayashi, M., Nikami, H., Morimatsu, M., and Saito, M. (1996) Neurosci. Lett. 213, 103-106[CrossRef][Medline] [Order article via Infotrieve] |
46. | Leloup, C., Arluison, M., Kassis, N., Lepetit, N., Cartier, N., Ferre, P., and Penicaud, L. (1996) Mol. Brain Res. 38, 45-53[CrossRef][Medline] [Order article via Infotrieve] |
47. | McCall, A. L., van Bueren, A. M., Huang, L., Stenbit, A., Celnik, E., and Charron, M. J. (1997) Brain Res. 744, 318-326[CrossRef][Medline] [Order article via Infotrieve] |
48. | Vannucci, S. J., Koehler-Stec, E. M., Li, K., Reynolds, T. H., Clark, R., and Simpson, I. A. (1998) Brain Res. 797, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
49. | Apelt, J., Mehlhorn, G., and Schliebs, R. (1999) J. Neurosci. Res. 57, 693-705[CrossRef][Medline] [Order article via Infotrieve] |
50. | Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, M. (1990) Cell 62, 317-329[Medline] [Order article via Infotrieve] |
51. | Haney, P. M., Slot, J. W., Piper, R. C., James, D. E., and Mueckler, M. (1991) J. Cell Biol. 114, 689-699[Abstract] |
52. | Courtoy, P. J., Quintart, J., Draye, J. P., and Baudhuin, P. (1988) Prog. Clin. Biol. Res. 270, 169-183[Medline] [Order article via Infotrieve] |
53. | Uphues, I., Kolter, T., Goud, B., and Eckel, J. (1994) Biochem. J. 301, 177-182[Medline] [Order article via Infotrieve] |
54. | Aledo, J. C., Darakhhan, F., and Hundal, H. S. (1995) Biochem. Biophys. Res. Commun. 215, 321-328[CrossRef][Medline] [Order article via Infotrieve] |
55. | Cormont, M., Bortoluzzi, M. N., Gautier, N., Mari, M., van Obberghen, E., and Le Marchand-Brustel, Y. (1996) Mol. Cell. Biol. 16, 6879-6886[Abstract] |
56. |
Vollenweider, P.,
Martin, S. S.,
Haruta, T.,
Morris, A. J.,
Nelson, J. G.,
Cormont, M.,
Le Marchand-Brustel, Y.,
Rose, D. W.,
and Olefsky, J. M.
(1997)
Endocrinology.
138,
4941-4949 |
57. |
Ralston, E.,
and Plough, T.
(1996)
J. Cell Sci.
109,
2967-2978 |
58. |
Garza, L. A.,
and Birnbaum, M. J.
(2000)
J. Biol. Chem.
275,
2560-2567 |
59. |
Thurmond, D. C.,
and Pessin, J. E.
(2000)
EMBO J.
19,
3565-3575 |