From the
Metabolic labeling and immunoprecipitation were used to analyze
the glucose-dependent regulation of GLUT1 synthesis, processing, and
turnover in a murine adipocyte cell line. Metabolically labeled GLUT1
from control cells migrated as a 46-kDa protein, while GLUT1 from cells
deprived of glucose for more than 12 h migrated as a 37-kDa protein. On
the basis of tunicamycin sensitivity, both GLUT1 species arose from a
common protein migrating at 36 kDa. In addition, the rate of synthesis
of GLUT1 in control and glucose-deprived cells was similar. In short
pulse-chase experiments, we distinguished two species arising from the
core GLUT1 protein in control cells; an intermediate and the mature
46-kDa species. In contrast, only one glycoform, the 37-kDa species,
arose from the core protein in glucose-deprived cells, which was not
further processed in either the presence or absence of glucose.
Although 12-18 h of glucose deprivation were required to affect
GLUT1 glycosylation, glucose-deprived cells quickly recovered the
ability to correctly glycosylate GLUT1 upon the readdition of glucose
(t <1 h). GLUT1 in control adipocytes exhibited a half-life
of approximately 14 h, while that in glucose-deprived adipocytes was
greater than 50 h. This effect was readily reversed upon the readdition
of glucose. In total, these data show that glucose deprivation alters
both the processing (glycosylation) and turnover (degradation) of
GLUT1. These results are discussed in light of transport function.
The transport of glucose across the plasma membrane is
accomplished by a family of integral membrane glycoproteins termed GLUT
proteins
(1) . The regulation of these proteins plays a key role
in the metabolism of sugar. In particular, the glucose-dependent
regulation of glucose transport has been described in many cell types
(for review, see Ref. 2). In each of these cell types, the result of
glucose deprivation is an increase in the maximal velocity of glucose
transport. However, the mechanism(s) (i.e. transcriptional,
post-transcriptional, and post-translational) responsible for this
stimulation vary according to cell type. Several investigators have
postulated that one component might be an alteration of transporter
turnover, particularly
GLUT1
(3, 4, 5, 6, 7, 8, 9, 10) .
Few studies, however, have investigated the processing and turnover of
GLUT1 directly because of the technical difficulties
involved
(11) . Thus our primary goal was to define the kinetics
of GLUT1 turnover.
In 3T3-L1 adipocytes, glucose deprivation results
in no significant change in the level of GLUT1 protein (as measured by
Western blotting) over the period in which transport induction is
observed
(12) . Beyond this time, we and others have reported the
accumulation of a lower molecular weight GLUT1 species which results in
a 2-fold increase in the total GLUT1 mass by 48 h of glucose
deprivation
(9, 12, 13) . We have previously
investigated the role of this second GLUT1 protein in transport
stimulation and provided evidence that it is not responsible for the
activation of transport during glucose deprivation
(12) . The
origin and characteristics of this lower molecular weight GLUT1 and its
relationship to the normal GLUT1 glycoform remain unclear. It is
possible that the new GLUT1 glycoform is a normal intermediate in GLUT1
processing that accumulates during glucose deprivation. Alternatively,
the new glycoform could contain an aberrant oligosaccharide generated
in the absence of glucose. To address these issues, we modified
existing methods of GLUT1 immunoprecipitation to improve recovery and
reduce nonspecific interactions. This has allowed us to clearly model
the processing and turnover of the metabolically labeled transporter in
normal and glucose-deprived adipocytes.
The GLUT1 glycoform in glucose-deprived
3T3-L1 adipocytes is interesting from several perspectives. First, it
is intriguing that over 12 h are required for its appearance. This
indicates that sufficient sugar is available to support normal
glycosylation for this period of glucose deprivation. Yet, it is
unlikely that any ``free'' sugar would be available to
support core oligosaccharide biosynthesis due to the high rate of
glucose utilization.
The oligosaccharide
structure on p37 form is currently unknown, although it is evidently
not the same as that on GLUT1 from LEC1 cells based on endoglycosidase
H sensitivity. However, previous studies provide some clues as to the
type of oligosaccharide that might be generated in the absence of
glucose
(20) . In CHO cells deprived of glucose, the synthesis of
lipid-linked oligosaccharide shifts from a normal
Glc
The function and targeting of p37 remains to be
determined. Studies directed toward understanding the role of
glycosylation in transport activity have provided ambiguous results.
Cells expressing a mutant GLUT1 transporter missing the glycosylation
site, Asn
Finally two
previous reports attempting to measure the half-life of GLUT1 by direct
methods have proven contradictory. Haspel et al.(11) observed a half-life of 90 min for GLUT1 in 3T3-L1 cells.
In contrast, Sargeant and Paquet
(24) studied the turnover of
GLUT1 in these cells and observed a half-life of approximately 19 h.
Our data showing a half-life of 14 h is more consistent with the
latter. In neither of these earlier papers was the effect of glucose
deprivation measured. Thus our study describes an aspect of GLUT1
regulation not previously identified. Even though glucose deprivation
had no specific effect on the rate of GLUT1 synthesis (despite the
difference in glycosylation), the effect on GLUT1 turnover was
striking. Clearly, the degradation of p37 was significantly inhibited.
Interestingly, so was the turnover of the total pool of membrane
protein. P46 turnover is affected only after 12 h of glucose
deprivation concurrent with the onset of abnormal or deficient
glycosylation as indicated by the appearance of p37. Thus, the loss of
glucose itself is not the signal for the inhibition of turnover.
Rather, the depletion of a specific glycoprotein, required for
degradation, may impair turnover. This is supported by that fact that
glucose readdition quickly restores normal GLUT1 glycosylation and
turnover.
Materials
DMEM(
)
was
obtained from Life Technologies, Inc. Glucose-free DMEM was prepared
exactly as commercially available except for the omission of glucose.
Calf serum (no. 1100-90) and fetal bovine serum (no. 1020-75) were
obtained from Intergen. Glucose-free fetal bovine serum was prepared by
dialyzing serum against PBS, pH 7.4, for 48 h at 4 °C, with a
molecular mass cutoff of 13,000 daltons. Tran
S-label (1100
Ci/mmol) was obtained from ICN. Polyoxyethylene 9 lauryl ether
(C
E
), octanoic acid, and anti-rabbit
IgG-conjugated horseradish peroxidase was obtained from Sigma. All
other reagents were of the highest quality commercially available.
Cell Culture
3T3-L1 fibroblasts were cultured and
differentiated as described previously
(14) . All other cell
culture procedures, including glucose deprivation, have been described
previously
(12) . 3T3-L1 adipocytes cultured in 100-mm plates (12
10
cells) were used for all immunoprecipitations.
Antibody Production and Purification
A polyclonal
antiserum, designated GT1, was generated to a peptide corresponding to
the COOH terminus of GLUT1 (CEELFHPLGADSQV) conjugated to keyhole
limpet hemocyanin as described previously
(15) . Total IgG was
purified from rabbit serum as described previously
(16) except
that the final IgG was dialyzed against PBS. Two mg of the GT1 peptide
were coupled to a Sulfolink matrix via the cysteine residue following
the manufacturer's instructions (Pierce). Fifteen mg of total IgG
were incubated with the peptide column and rotated end-over-end at room
temperature for 2 h. Unbound IgG was washed from the column with PBS.
Specifically bound IgG was eluted with 0.1 M glycine, pH 3.0,
and neutralized with 0.1 ml 1 M Tris base. Eluted IgG
fractions were pooled before being dialyzed against PBS for 12 h at 4
°C. The final IgG was stored at -20 °C as a 0.5 mg/ml
solution.
Metabolic Radiolabeling of 3T3-L1
Adipocytes
3T3-L1 adipocytes were incubated in 8 ml of
methionine- and cysteine-free DMEM without added serum for 1 h to
deplete intracellular pools. The depletion medium was then removed, and
the cells were incubated in 2 ml of methionine- and cysteine-free DMEM
containing 200-500 µCi/ml TranS-label (as noted
in the figure legends) for the specific times indicated (10-180
min). For chase periods, the labeling medium was aspirated and replaced
with 8 ml complete DMEM, 10% fetal bovine serum containing 2
mM methionine and 2 mM cysteine. These conditions
were established to generate an adequate signal for immunoprecipitation
while minimizing methionine and cysteine depletion based on previous
reports of alterations in protein turnover with prolonged amino acid
depletion (11).
Immunoprecipitation
For GLUT1 immunoprecipitation,
we utilized a procedure described previously
(17) , with the
following modifications. Total membrane fractions, prepared as
described previously
(12) , were homogenized with 20 strokes in 1
ml of extraction buffer (PBS containing 1 mM EDTA, 2%
CE
, 0.1% SDS, and protease inhibitor (20
µg/ml each of aprotinin, leupeptin, pepstatin, tosylphenylalanyl
chloromethyl ketone,
N
-p-tosyl-L-lysine
chloromethyl ketone, and 1 mM phenylmethylsulfonyl fluoride).
Insoluble material was sedimented in a microcentrifuge for 5 min at 4
°C. The supernatant was recovered, frozen in liquid nitrogen, and
stored at -20 °C. Thawed extracts (1.5-2 mg) were
pre-cleared with 5 µl of an unrelated nonimmune antiserum and
collected with 25 µl of a 50% suspension of protein A-Sepharose for
1 h at 4 °C. Samples were spun briefly in a microcentrifuge, and
pre-cleared supernatants were transferred to new tubes. Extracts were
adjusted to equal protein concentration and incubated with 5 µg of
anti-GT1 antibody for 3 h at 4 °C with mixing. Twenty-five µl
of the protein A-Sepharose suspension was then added for 2 h to collect
the immunoprecipitates. The protein A-Sepharose was then washed three
times with extraction buffer, followed by four times for 10 min with
extraction buffer containing 1 M NaCl. Immunoprecipitates were
released by incubation in 0.1 ml of sample dilution buffer containing 6
M urea and 10%
-mercaptoethanol for 15 min at 37 °C.
The supernatants were loaded onto an 8% SDS-PAGE gel and run overnight
at 50 V. For fluorography, the gels were fixed in 10% trichloroacetic
acid, 40% MeOH for 30 min, soaked in water for 30 min, and then soaked
in 1 M sodium salicylate for 1 h before drying at 60 °C
under vacuum. Dried gels were exposed to Amersham Hyperfilm typically
for 4 days.
Endoglycosidase Digestions
Twenty µg of
membrane protein from control or glucose-deprived adipocytes and 10
µg from LEC1 CHO cells were denatured in 50 mM
-mercaptoethanol, 0.5% SDS for 20 min at 37 °C. Then, for
N-glycosidase F digestions, the samples were brought to 150
mM Tris-HCl, pH 8.0, 1% Nonidet P-40, protease inhibitors as
above, 1.25 units of N-glycosidase F and then incubated at 37
°C for 2 h. For endoglycosidase H digestions, the denatured protein
sample was brought to 75 mM sodium citrate, pH 5.5, protease
inhibitors as above, and 2.5 milliunits of endoglycosidase H and
incubated at 37 °C for 2 h. An equal volume of 2
sample
dilution buffer was added and loaded onto an 8% SDS-PAGE gel for
separation.
Electrotransfer and Western Blotting
Transfer of
proteins to nitrocellulose and Western analysis were performed as
described previously
(12) .
RESULTS
Specificity and Efficiency of GLUT1
Immunoprecipitation
The GLUT1 immunoprecipitation procedure was
assessed using metabolically labeled control or glucose-deprived
adipocytes. Fig. 1shows a fluorograph of the immunoprecipitates.
A wide band migrating at 46 kDa (p46) was immunoprecipitated
from membrane extracts of control cells using purified anti-GT1
antibody (-GT1, + glucose). In contrast, a
37-kDa protein was immunoprecipitated from cells deprived of glucose
for 36 h (-GT1, -glucose). The
specificity of the immunoprecipitation procedure was verified by
immunoprecipitating GLUT1 in the presence of 1 µg of the GT1
peptide. Inclusion of this peptide completely blocked the
immunoprecipitation of p46 GLUT1 in control cells, as well as p37 GLUT1
in glucose-deprived cells (+GT1, ±
glucose). In a similar competition experiment, a GLUT4
carboxyl-terminal peptide (CSTELEYLGPDEND) did not block the
immunoprecipitation of GLUT1 (+GT4, ±
glucose). GLUT1 protein of either molecular mass was not
observed in membrane extracts immunoprecipitated with protein
A-Sepharose alone, with preimmune serum, or with 5 µg of IgG from
an unrelated antisera (-Ab, PIS, IRR,
± glucose). GLUT1 protein was also not observed when
extracts were immunoprecipitated with an equal amount of the unbound
antibody fraction (flow-through) from peptide purification of the same
antisera, indicating that the purification step removed the majority of
the GLUT1 specific IgG (FT, ± glucose).
Figure 1:
Specificity and
efficiency of GLUT1 immunoprecipitation. Cells, cultured with or
without glucose for 36 h, were metabolically labeled with 200
µCi/ml TranS-label for 1 h as described under
``Experimental Procedures.'' GLUT1 was immunoprecipitated
from a total membrane fraction as indicated, separated by SDS-PAGE, and
visualized by fluorography. -Ab, no antibody;
PIS, preimmune serum; IRR, unrelated serum;
-GT1, immunoprecipitated in the absence of peptide;
+GT1, inclusion of 1 µg of GT1 peptide;
+GT4, inclusion of 1 µg of GT4 peptide; FT,
unbound antibody fraction from peptide
purification.
We
determined the efficiency of the immunoprecipitation protocol by
sequential rounds of immunoprecipitation with the anti-GT1 antibody.
Approximately 80% of GLUT1 was collected during the first round of
immunoprecipitation in either control or glucose-deprived cells. The
remainder was recovered during a second round of immunoprecipitation
(data not shown). In the experiments described herein, only one round
of immunoprecipitation was used.
Synthesis and Glycosylation of GLUT1 in Control and
Glucose-deprived 3T3-L1 Adipocytes
To determine the role of
glucose in regulating the synthetic rate of GLUT1, control and
glucose-deprived adipocytes were metabolically labeled for 10-180
min. Fig. 2shows a fluorograph of the GLUT1 immunoprecipitates.
Two GLUT1 species were synthesized in control cells during the first 10
min of labeling, a 36-kDa protein and a glycosylated intermediate
migrating at 42 kDa. The 36-kDa protein is very likely the core protein
based on the predicted molecular weight of GLUT1 in the absence of
oligosaccharide and tunicamycin experiments (see below). With 20 min of
labeling, the mature 46-kDa GLUT1 was observed along with p36 and p42.
In glucose-deprived cells labeled for 10 min, only the 37-kDa GLUT1
protein was observed. The 36-kDa core GLUT1 protein is likely obscured
due to the width of the p37 protein. Lighter exposures provided no
further discrimination. No additional GLUT1 species were synthesized
during the remainder of labeling period. Although the intensity of
total GLUT1 protein in glucose-deprived cells was greater than that of
control cells (Fig. 2B), this effect was not specific to
GLUT1 when taking into account an equal increase in radioactivity of
the membrane fraction from glucose-deprived cells
(Fig. 2B, inset). This phenomenon likely
represents an increase in the specific activity of the methionine and
cysteine pools in glucose-deprived cells. Taken together, these data
demonstrate that the synthetic rate of GLUT1 was unaffected by glucose
availability despite the differences in glycosylation.
Figure 2:
GLUT1 synthesis in control and
glucose-deprived adipocytes. Panel A, cells, cultured with or
without glucose for 36 h, were metabolically labeled with 200
µCi/ml TranS-label for the times indicated as
described under ``Experimental Procedures.'' GLUT1 was then
immunoprecipitated from membrane extracts and analyzed by SDS-PAGE and
fluorography. Panel B, densitometry of total GLUT1 during
labeling. Total GLUT1 intensity was obtained in all experiments by
summing the appropriate bands after densitometry of each species
separately (i.e.p36 + p42 +
p46). Inset, radioactivity in membrane fraction
during labeling relative to 10 min label. This result is representative
of at least two independent experiments.
To confirm
the hypothesis that p37 GLUT1 arises from post-translational
modification with N-linked oligosaccharide, control and
glucose-deprived adipocytes were incubated in the presence or absence
of tunicamycin before labeling. In the absence of tunicamycin, the
synthesis of the 46- and 37-kDa GLUT1 species was observed in control
and glucose-deprived cells, respectively, as expected (Fig. 3).
In contrast, GLUT1 immunoprecipitated from tunicamycin-treated cells
migrated at 36 kDa regardless of metabolic state. This demonstrates
that both p46 and p37 arise from the core protein and that both species
are glycosylated, albeit to different extents.
Figure 3:
Glycosylation of GLUT1 in control and
glucose-deprived adipocytes. Cells, cultured with or without glucose
for 36 h, were incubated in the presence (+) or absence (-)
of tunicamycin (2.5 µg/ml) for 24 h before being metabolically
labeled with 200 µCi/ml TranS-label for 1 h as
described under ``Experimental Procedures.'' GLUT1 was then
immunoprecipitated from membrane extracts and visualized by SDS-PAGE
and fluorography. This result is representative of at least two
independent experiments.
Regulation of GLUT1 Processing by Glucose
To
examine the processing pathway of GLUT1 in control and glucose-deprived
adipocytes, cells were metabolically labeled for 10 min and then chased
for a total of 60 min. In control cells, the 36- and 42-kDa GLUT1
precursors synthesized during the pulse disappeared followed by the
emergence of the mature GLUT1 at 46 kDa after 20 min of chase
(Fig. 4A). In contrast, p37 synthesized in
glucose-deprived cells was unchanged when chased in the absence of
glucose (Fig. 4B). Although this indicated that p37 was
not processed in glucose-deprived cells, it did not rule out the
possibility that p37 could be processed if glucose was
present. We therefore tested this hypothesis and found that when
glucose-deprived cells were labeled for 10 min and then chased in the
presence of glucose, p37 remained unchanged (Fig. 4C).
These results demonstrate that although 3T3-L1 adipocytes have the
capacity to glycosylate GLUT1 to some extent in the absence of glucose,
the transporter cannot be further processed due to either the structure
of the oligosaccharide or the location of GLUT1 along the processing
pathway.
Figure 4:
Processing of GLUT1 in control and
glucose-deprived adipocytes. Cells incubated with
(+glucose) or without (-glucose) glucose
for 36 h were labeled with 500 µCi/ml TranS-label for
10 min and then chased for a total of 60 min. GLUT1 was
immunoprecipitated from membrane extracts of each and analyzed by
SDS-PAGE and fluorography. Panel A, control adipocytes chased
in the presence of glucose. Panel B, glucose-deprived
adipocytes chased in the absence of glucose. Panel C,
glucose-deprived cells chased in the presence of glucose. Shown is the
result of a single experiment.
Synthesis of GLUT1 during Glucose Deprivation and
Refeeding
To determine the time required for glucose deprivation
to affect GLUT1 glycosylation, control cells were placed into
glucose-free medium and labeled for 1 h at specific times during the
next 36 h. Despite glucose withdrawal, p46 was the only GLUT1 glycoform
synthesized during the first 12 h of glucose deprivation
(Fig. 5A). Beyond 12 h of glucose deprivation, the major
GLUT1 glycoform synthesized shifted to p37. Densitometry of the GLUT1
bands indicated that the total amount of GLUT1 in glucose-deprived
cells remained nearly constant (Fig. 5B). To examine the
reversibility of this effect, cells deprived of glucose for 36 h were
placed into medium containing glucose and labeled for 1 h during
refeeding. As expected, p37 was the only form synthesized in the
glucose-deprived cells. Within 6 h of refeeding, however, only p46 was
synthesized (Fig. 5C). Further, the readdition of
glucose resulted in a 3.5-fold increase in GLUT1 despite only a 40%
increase in total membrane radioactivity (Fig. 5D). To
examine this recovery in more detail, we immunoprecipitated GLUT1 from
adipocytes labeled for 1 h at each hour during the first 6 h of
refeeding. Surprisingly, within 1 h of glucose readdition, only the
normal p46 glycoform was synthesized; no metabolically labeled p37 was
observed (data not shown).
Figure 5:
Synthesis of GLUT1 during glucose
deprivation and refeeding. Panel A, control cells were placed
into glucose-free medium and labeled for 1 h with 200 µCi/ml
TranS-label at the times indicated. GLUT1 was
immunoprecipitated from membrane extracts and analyzed by SDS-PAGE and
fluorography. Panel B, densitometry of total GLUT1 during
glucose deprivation. Panel C, glucose-deprived (36 h) cells
were placed into medium containing glucose and labeled for 1 h with 200
µCi/ml Tran
S-label at times indicated. GLUT1 was then
analyzed as in (A). Panel D, densitometry of total
GLUT1 during glucose refeeding. These results are representative of at
least three independent experiments.
GLUT1 Half-life in Control and Glucose-deprived
Adipocytes
To determine the glucose-dependent regulation of
GLUT1 turnover in 3T3-L1 adipocytes, control and glucose-deprived cells
were metabolically labeled and then chased in either the presence or
absence of glucose. GLUT1 (p46) in control adipocytes exhibited a
half-life of approximately 14 h (Fig. 6, A and
B). GLUT1 from control adipocytes chased in the absence of
glucose exhibited a similar turnover until 12 h of glucose deprivation.
After this time, the degradation of GLUT1 was significantly reduced.
GLUT1 (p37) synthesized in glucose-deprived cells was not significantly
degraded in the absence of glucose (Fig. 6, C and
D). Although not present during the initial labeling (time
= 0), a second 44-kDa glycoform appeared during the chase, whose
turnover time was identical to that of p37. In glucose-deprived
adipocytes chased in medium containing glucose, the turnover of GLUT1
(both p44 and p37 glycoforms) was similar to that of p46 in control
adipocytes.
Figure 6:
Turnover of GLUT1 in control,
glucose-deprived, and refed adipocytes. Control or glucose-deprived
cells were labeled with 200 µCi/ml TranS-label for 1 h
and then chased in the presence (+) or absence (-) of
glucose. GLUT1 was immunoprecipitated from membrane extracts and
analyzed by SDS-PAGE and fluorography. Panel A, GLUT1
immunoprecipitates from control cells chased in the presence or absence
of glucose. Panel B, Densitometry of total GLUT1 during the
chase period from Panel A. Panel C, GLUT1 immunoprecipitates
from glucose deprived cells chased in the presence or absence of
glucose. Panel D, Densitometry of total GLUT1 during the chase
period from Panel C. Results shown are from one experiment
which is representative of at least four independent
experiments.
Glycosidase Digestion of GLUT1 Glycoforms
In order
to gain insight into the type of oligosaccharide structure attached to
p37, we compared its mobility and sensitivity to endoglycosidases with
GLUT1 from LEC1 CHO cells, a cell line which lacks
N-acetylglucosaminyl transferase I activity
(18) . Thus,
total membrane protein from control adipocytes, glucose-deprived
adipocytes, and LEC1 CHO cells were treated with or without
N-glycosidase F, which removes N-linked
oligosaccharides leaving core protein, or endoglycosidase H, which
specifically cleaves oligosaccharides with high mannose structure. As
expected, the normal GLUT1 glycoform (p46) which contains a complex
oligosaccharide was sensitive to digestion by N-glycosidase F
but not endoglycosidase H as assessed by Western blotting
(Fig. 7). The mobility of GLUT1 from LEC1 CHO cells was
significantly faster than p46, a consequence of the inability of these
cells to process glycoproteins beyond a high mannose structure. Thus,
GLUT1 in these cells was sensitive to both N-glycosidase F and
endoglycosidase H. In glucose-deprived adipocytes, both p46 and p37
were observed by Western blotting and both were sensitive to
N-glycosidase F treatment. The migration of p37 GLUT1 was not
altered by treatment with endoglycosidase H, indicating that it does
not contain an oligosaccharide similar to GLUT1 from LEC1 CHO cells
despite their similar migration.
Figure 7:
Endoglycosidase digestion of GLUT1
glycoforms from 3T3-L1 adipocytes and LEC1 CHO. Membrane protein from
control and glucose-deprived 3T3-L1 adipocytes (20 µg) and from
LEC1 CHO cells (10 µg) was denatured and treated with
endoglycosidase as described under ``Experimental
Procedures.'' Membrane proteins were then separated by SDS-PAGE
and transferred to nitrocellulose. GLUT1 was then detected by Western
analysis and visualized by enhanced chemiluminesence. Results shown are
representative of at least four independent
experiments.
DISCUSSION
In this report, we used immunoprecipitation to analyze the
effect of glucose deprivation on the biosynthesis, processing, and
turnover of GLUT1 in 3T3-L1 adipocytes. Although several studies have
reported the appearance of a low molecular weight form of GLUT1
resulting from the withdrawal of glucose, the relationship, if any,
between this form and the normal molecular weight species has not been
investigated. Thus we have extended these prior studies in several
ways. First, our work demonstrated that p37 GLUT1 is a newly
synthesized protein rather than a breakdown product of the normal
transporter. Second, we determined that p37 is not a precursor to the
normal form of GLUT1 (p46). Finally, we demonstrated that p37 is
post-translationally processed in 3T3-L1 adipocytes by a pathway that
is unique from that of p46 resulting in an abbreviated and likely
aberrant oligosaccharide. These conclusions are based on the following
observations. In control cells, we identified a single mature form of
GLUT1 that migrated as a broad band at approximately 46 kDa. Two
precursor forms were distinguished from the mature GLUT1. These
represent the core protein (36 kDa) and an intermediate (42 kDa) in the
processing pathway consistent with analysis of GLUT1 processing in a
cell-free system
(19) . When metabolically labeled GLUT1 was
immunoprecipitated from glucose-deprived cells, we observed a 37-kDa
protein. In the presence of tunicamycin, only one form of GLUT1 (p36)
was synthesized in either control or glucose-deprived cells, indicating
that the core protein was not altered by glucose deprivation. This also
indicates that p37, like the normal GLUT1 glycoform, arose from the
36-kDa core protein by modification with an N-linked
oligosaccharide. It should be noted that not all cells appear competent
to glycosylate GLUT1 in the absence of glucose. The lower molecular
mass GLUT1 observed in glucose-deprived rat kidney cells, for example,
migrates at the same molecular weight as GLUT1 from these cells
incubated with tunicamycin or GLUT1 transporter isolated from these
cells treated with endoglycosidase
(9) . Glycosylation therefore
represents another cell type-specific aspect of the regulation of GLUT1
expression by glucose.
(
)
However, glycogen
breakdown may provide sufficient substrate for this process during the
early phase of deprivation. Only when the glycogen pool is depleted
would p37 accumulate. Yet, in the face of extended glucose deprivation
with no apparent source of glucose for oligosaccharide synthesis, GLUT1
(p37) is still glycosylated, albeit in abbreviated form. It is possible
that carbohydrate is scavenged from the degradation of other
glycoproteins whose function is not needed under these conditions.
Alternatively, the lipid-linked oligosaccharide pool may not be
completely depleted between 12 and 36 h of deprivation and thus
provides continuous core oligosaccharide.
-Man
GlcNAc
to an alternative
structure, Glc
Man
GlcNAc
. The
transfer of this oligosaccharide to protein acceptors occurred
normally, although the resulting glycoproteins were endoglycosidase H
insensitive. It could therefore be hypothesized that 3T3-L1 adipocytes
might also generate a similar oligosaccharide in the absence of
glucose, resulting in a GLUT1 protein that was both slightly greater in
molecular weight than the core protein and insensitive to
endoglycosidase H. Although p37 GLUT1 displays these characteristics,
confirmation of this hypothesis would require purification of the
transporter in significant quantities and subsequent carbohydrate
sequencing.
, retained some glucose transport
activity
(21) . However, treatment of GLUT1-containing vesicles
with exo- and endoglycosidases resulted in a loss of transport
activity
(22) . Likewise, the role of glycosylation in targeting
GLUT1 to the functional compartment (i.e. plasma membrane) is
not clear. The mature GLUT1 transporter in 3T3-L1 adipocytes and other
cell types at the cell surface is a complex-type glycoprotein. However,
GLUT1 from the LEC1 CHO cell line resides at the cell surface as
evidenced by the ability of these cells to transport
glucose
(23) . Clearly, the type of oligosaccharide present or
endoglycosidase sensitivity alone cannot be used to predict or define
protein localization. The glucose deprivation model, coupled with
subcellular fractionation, may provide information regarding the
effects of alternative glycosylation on GLUT1 targeting.
E
, polyoxyethylene
9 lauryl ether.
cells/h (H.
H. Kitzman, R. J. McMahon, P. M. Fadia, and S. C. Frost, submitted for
publication).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.