THE JOURNAL OF BIOLOGICAL CHEMISTRY
5929 –5940, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycolysis and Glutamate Accumulation into Synaptic Vesicles
ROLE OF GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE AND 3-PHOSPHOGLYCERATE KINASE*
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211617200
Atsushi Ikemoto‡§, David G. Bole‡, and Tetsufumi Ueda‡¶储**
From the ‡Mental Health Research Institute, Departments of ¶Pharmacology and 储Psychiatry,
University of Michigan Medical School, Ann Arbor, Michigan 48109-0669
Glucose is the major source of brain energy and is
essential for maintaining normal brain and neuronal
function. Hypoglycemia causes impaired synaptic trans-
mission. This occurs even before significant reduction
in global cellular ATP concentration, and relationships
among glycolysis, ATP supply, and synaptic transmis-
sion are not well understood. We demonstrate that the
glycolytic enzymes glyceraldehyde phosphate dehydro-
genase (GAPDH) and 3-phosphoglycerate kinase
(3-PGK) are enriched in synaptic vesicles, forming a
functional complex, and that synaptic vesicles are capa-
ble of accumulating the excitatory neurotransmitter
glutamate by harnessing ATP produced by vesicle-
bound GAPDH/3-PGK at the expense of their substrates.
The GAPDH inhibitor iodoacetate suppressed GAPDH/
3-PGK-dependent, but not exogenous ATP-dependent,
[3H]glutamate uptake into isolated synaptic vesicles. It
also decreased vesicular [3H]glutamate content in the
nerve ending preparation synaptosome; this decrease
was reflected in reduction of depolarization-induced
[3H]glutamate release. In contrast, oligomycin, a mito-
chondrial ATP synthase inhibitor, had minimal effect on
any of these parameters. ADP at concentrations above
0.1 mM inhibited vesicular glutamate and dissipated
membrane potential. This suggests that the coupled
GAPDH/3-PGK system, which converts ADP to ATP, en-
sures maximal glutamate accumulation into presynap-
tic vesicles. Together, these observations provide in-
sight into the essential nature of glycolysis in sustaining
normal synaptic transmission.
Glycolysis plays a vital role in maintaining normal brain
function. Glucose is known to serve as the major substrate for
cerebral energy under normal conditions (1). Recent evidence
suggests a direct correlation between glucose utilization and
cognitive function (2). Reduction of glucose levels results in
pathophysiological states and abnormal electrophysiological
activity; however, this occurs long before significant alteration
in tissue ATP levels is detected (3–7). Substitution of pyruvate
for glucose does not support normal evoked neuronal activity,
although tissue ATP level returns to normal (8 –10). Abnormal
* This work was supported in part by National Institutes of Health
Grants NS 24384, NS 36656, and NS 42200 and a grant from Taisho
Pharmaceutical Co., Ltd. (Tokyo, Japan). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ On leave from the Dept. of Biological Chemistry, Faculty of Phar-
maceutical Sciences, Nagoya City University, 467-8603 Nagoya, Japan.
** To whom correspondence should be addressed: Mental Health
Research Institute at MSRB II, C570D, The University of Michigan,
1150 W. Medical Center Dr., Ann Arbor, MI 48109-0669. Tel.: 734-763-
3790; Fax: 734-936-2690; E-mail: tueda@umich.edu.
This paper is available on line at http://www.jbc.org
Vol. 278, No. 8, Issue of February 21, pp.
Printed in U.S.A.
Received for publication, November 14, 2002
synaptic transmission caused by hypoglycemia occurs in part if
not entirely by a presynaptic mechanism (7, 11, 12). Fleck et al.
(7) have shown that substantial reduction of extracellular glu-
cose results in a decrease in stimulus-evoked Glu release, with
no changes in ATP levels. These studies together suggest that
glycolysis or glycolytic intermediate(s) are necessary for nor-
mal synaptic transmission independent of global cellular ATP
levels.
In an attempt to reveal the underlying mechanism of hypo-
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glycemia-induced aberrant synaptic transmission, we previ-
ously explored the possibility that glycolytic intermediates
could modify proteins localized in the nerve ending (13, 14).
3-Phosphoglycerate (3-PG)1 was demonstrated to stimulate
phosphorylation of 155- and 72-kDa proteins. The latter was
identified as glucose-1,6-bisphosphate synthetase, and 1,3-
bisphosphoglycerate (1,3-BPG) was found to serve as the direct
substrate for phosphorylation of this enzyme, by donating
1-phosphate. Both of these phosphorylated proteins are en-
riched in the synaptosomal (nerve ending preparation) as well
as cell body soluble fractions, but the significance of these
modifications in synaptic transmission remains unclear.
In this paper, we show that the glycolytic intermediate 1,3-
BPG forms an acyl-enzyme intermediate with vesicle-bound
glyceraldehyde phosphate dehydrogenase (GAPDH), that ves-
icle-bound GAPDH exists in a complex with 3-phosphoglycer-
ate kinase (3-PGK), and that activation of vesicle-associated
GAPDH and 3-PGK is sufficient to support vesicular uptake of
Glu. Glutamate is now recognized as the major excitatory neu-
rotransmitter responsible for triggering neuronal firing, in the
vertebrate central nervous system (15–23). As such, proper Glu
synaptic transmission is not only essential for basic neuronal
communication but also is involved in learning and memory
formation (19, 20). Glutamate accumulation into synaptic ves-
icles in the nerve terminal is an initial crucial step in Glu
transmission (18, 22–26). This process requires ATP to gener-
ate an electrochemical gradient, which is the driving force for
Glu uptake into synaptic vesicles (27–34).
We present evidence that glycolytically produced ATP, in
particular that produced by GAPDH and 3-PGK, but not
mitochondria-derived ATP, is harnessed for accumulation of
Glu into synaptic vesicles in synaptosomes; Glu transported
into synaptic vesicles in this manner is released upon depo-
larization. These findings could provide an explanation for
hypoglycemia-induced aberrant synaptic transmission and
1
The abbreviations used are: 3-PG, 3-phosphoglycerate; t-ACPD,
trans-1-aminocyclopentane-1,3-dicarboxylic acid; 4-AP, 4-aminopyri-
dine; 1,3-BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone
phosphate; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydra-
zone; GAP, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; 3-PGK, 3-phosphoglycerate kinase; PGM,
monophosphoglycerate mutase; ACSF, artificial cerebrospinal fluid.
5929
5930 GAPDH and Vesicular Glu Accumulation
insight into the essential nature of glycolysis in normal
synaptic transmission.

EXPERIMENTAL PROCEDURES
Materials—[␥-32P]ATP (6,000 Ci/mmol) was obtained from
PerkinElmer Life Sciences. L-[G-3H]Glutamic acid (42.0 Ci/mmol) was
purchased from Amersham Biosciences. The affinity-purified polyclonal
antibodies (rabbit IgG) specific to recombinant monophosphoglycerate
mutase (PGM) type B was kindly provided by Oriental Yeast Co., Ltd.
(Tokyo, Japan). Anti-GAPDH monoclonal antibody (mouse IgG) and
anti-3-PGK polyclonal antibody (rabbit IgG) were purchased from U.S.
Biological (Swampscott, MA) and Accurate Chemical & Science Co.
(Westbury, NY), respectively. Glycolytic enzymes and all other chemi-
cals were purchased from Sigma-Aldrich unless mentioned elsewhere.
Preparation of Subcellular Fractions—Synaptic vesicles were pre-
pared from bovine cerebrum through the discontinuous sucrose gradi-
ent procedure as described previously (35). The subcellular fractions of
bovine cerebrum were prepared as described previously (36). Synapto-
somes were prepared from cerebra of male Sprague-Dawley rats (150 –
200 g) and purified through the Percoll gradient centrifugation step, as
described by Dunkley et al. (37). Protein concentration was determined
by the method of Bradford (38) with a Coomassie protein assay reagent
kit (Pierce) with bovine serum albumin as standard protein.
Synthesis of [3-32P]1,3-BPG—[32P]Dihydroxyacetone phosphate
(DHAP) was prepared by phosphorylation of dihydroxyacetone by glyc-
erol kinase with [␥-32P]ATP in a mixture containing 5 mM Tris-HCl (pH

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7.0), 40 ␮M MgSO4, 10 mM dihydroxyacetone, 20 ␮M [␥-32P]ATP (6,000
Ci/mmol), and 0.5 units of glycerol kinase (Bacillus stearothermophi-
lus). The reaction was performed in a volume of 40 ␮l at 37 °C for 20
min. [3-32P]1,3-BPG was prepared from [32P]DHAP by conversion to
[32P]GAP with triose-phosphate isomerase, followed by a GAPDH reac-
tion in the presence of the lactate dehydrogenase-coupled NAD-regen-
erating system. The reaction mixture (400 ␮l) contained 12.5 mM tri-
ethanolamine (pH 8.0), 0.2 mM EDTA, 2 mM NAD, 2 mM sodium
pyruvate, 2 mM KH2PO4, 50 ␮M DHAP, 3.2 units of GAPDH (rabbit
muscle), 20 units of triose-phosphate isomerase (rabbit muscle), 10
units of lactate dehydrogenase (rabbit muscle), and 40 ␮l of the [32P]D-
HAP reaction mixture. The entire mixture was incubated at 25 °C for 5
min, filtered to remove the enzymes by an Amicon Centricon-10 con-
centrator at 4 °C, and put onto DEAE-cellulose (Whatman DE32, 1.2 ⫻
2.2 cm) previously equilibrated with 10 mM glycylglycine, pH 7.4. Elu-
tion was carried out with stepwise increases in the NaCl concentration:
50, 75, 100, 125, 150, and 200 mM. DHAP, GAP, and inorganic phos-
phate (Pi) were eluted with 75 mM NaCl in the same buffer. 3-PG and
1,3-BPG were eluted with 125 and 150 mM NaCl, respectively, in the
same buffer. All the compounds thus prepared were stored at ⫺80 °C
until use. FIG. 1. GAPDH is labeled with [3-32P]1,3-BPG and enriched in
Radioactive compounds were analyzed by high pressure liquid chro- synaptic vesicles. Synaptic vesicles were incubated at 37 °C for 10 s
matography on a Whatman Partisil 10 SAX WCS column (4.6 ⫻ 250 with [3-32P]1,3-BPG in the absence or presence of 4 mM MgSO4 (Mg2⫹)
mm), comparing their retention times with those of nonradioactive or 50 ␮M 3-PG and subjected to SDS-PAGE under neutral pH condi-
tions, according to the method of Fairbanks (A) or under alkaline pH
authentic standards monitored at 214 nm. The column was equilibrated
conditions (standard SDS-PAGE) according to the method of Laemmli
with 0.4 M sodium phosphate buffer (pH 3.2), and glycolytic intermedi-
(B), followed by autoradiography. C, the 0.2 M NaCl extract of the
ates and nucleotides were eluted isocratically, as described previously synaptic vesicle fraction was incubated at 37 °C for 10 s with [3-32P]1,3-
(14). Retention times for glycolytic intermediates were 4.1 min for BPG in the presence (anti-GAPDH, mouse IgG) or absence (anti-PGM,
DHAP and GAP, 5.3 min for Pi, 5.9 min for 2-phosphoglycerate and rabbit IgG) of 4 mM Mg2⫹; immunoprecipitated with the anti-GAPDH
3-PG, 6.0 min for ADP, 7.4 min for phosphoenolpyruvate, 15.4 min for antibody, anti-PGM antibody, mouse IgG (control for anti-GAPDH), or
1,3-BPG, and 30.0 min for ATP. rabbit IgG (control for anti-PGM); and subjected to SDS-PAGE under
Protein Labeling with [3-32P]1,3-BPG—The synaptic vesicle fraction neutral pH conditions, followed by autoradiography. D, various subcel-
(30 ␮g of protein) was preincubated at 37 °C for 30 s in 27 ␮l of 5 mM lular fractions (30 ␮g of protein) as indicated were incubated at 37 °C
Tris-maleate (pH 7.4). The reaction was initiated by the addition of 3 ␮l for 10 s with 140 nM [3-32P]1,3-BPG and subjected to SDS-PAGE under
of [3-32P]1,3-BPG (240 Ci/mmol) to a final concentration of 140 nM and neutral pH conditions, followed by autoradiography. E, the subcellular
allowed to continue for 10 s. For electrophoretic protein separation fractions were separated by standard SDS-PAGE, blotted onto polyvi-
under neutral pH conditions, the reaction was terminated by the addi- nylidene difluoride membranes, and detected with anti-GAPDH. F, the
tion of 10 ␮l of buffer containing 4% SDS, 30% sucrose, 40 mM Tris-HCl synaptic vesicle fraction was treated with various concentrations of
(pH 7.0), 4 mM EDTA, 160 mM 2-mercaptoethanol, and 50 ␮g/ml brom- NaCl, and the salt extracts (supernatant (Sup)) and the salt-insoluble
phenol blue; aliquots (25 ␮l) were subjected to polyacrylamide gel (1% material (Pellet) were analyzed for GAPDH as described above. The
data shown are representative of results obtained from three separate
SDS and 5.6% acrylamide) without stacking gel according to the method
experiments. Syn. cytosol, synaptosomal cytosol; Per. cytosol, perikaryal
of Fairbanks et al. (39). For electrophoretic protein separation under
cytosol (cell body cytosol); Pl. membrane, plasma membrane.
alkaline pH conditions (standard SDS-PAGE), the reaction was termi-
nated by the addition of 10 ␮l of SDS sample buffer containing 4% SDS,
40% glycerol, 0.2 M Tris-HCl (pH 6.8), 20% 2-mercaptoethanol, and 50 bilized protein G (0.1 ml as 50% slurry) and chemically cross-linked,
␮g/ml bromphenol blue; aliquots (25 ␮l) were subjected to polyacryl- using the Seize X mammalian immunoprecipitation kit (Pierce). The
amide gel (0.1% SDS and 12% acrylamide) according to the method of synaptic vesicle fraction (50 ␮g of protein) was stirred in 0.2 ml of buffer
Laemmli (40), except for omission of sample boiling. Autoradiography containing 0.32 M sucrose, 4 mM Tris-maleate (pH 7.4), and 0.2 M NaCl
was carried out as described previously (41) and analyzed using an for 5 min at 4 °C and centrifuged at 200,000 gmax for 1 h at 4 °C. When
32
image analyzer (Bio-Rad Gel Doc 2000). P-labeled protein was detected, the synaptic vesicle fraction was in-
Immunoprecipitation—Antibodies (10 ␮g) were absorbed onto immo- cubated at 37 °C for 10 s with the same buffer containing 140 nM (240
 GAPDH and Vesicular Glu Accumulation
FIG. 2. Association of 3-PGK with synaptic vesicles mediated
by GAPDH. A, various subcellular fractions as indicated were ana-
lyzed for 3-PGK by SDS-PAGE/Western blot as described in the Fig. 1
legend, except for use of anti-3-PGK antibodies instead of anti-GAPDH
antibody. B, the synaptic vesicle fraction was treated with various
concentrations of NaCl as indicated, and the salt extracts (supernatant
(Sup)) and the salt-insoluble material (Pellet) were analyzed for 3-PGK
by SDS-PAGE/Western blotting probed with anti-3-PGK antibodies.
The 0.2 M NaCl extract of synaptic vesicles was subjected to immuno-
precipitation with rabbit IgG or anti-3-PGK antibodies (C) and with
mouse IgG or the anti-GAPDH antibody (D); the immunoprecipitates
were analyzed for GAPDH (C) and 3-PGK (D), respectively. E, synaptic
vesicles (SV) were washed twice with 0.8 M NaCl and incubated in the
absence or presence of GAPDH, each with or without 3-PGK at 37 °C for
10 min, followed by two additional washings with low salt buffer. The
synaptic vesicle pellets were then analyzed for GAPDH and 3-PGK. The
data shown are representative of results obtained from three separate
experiments.
Ci/mmol) [3-32P]1,3-BPG. The supernatant (180 ␮l) was subjected to
immunoprecipitation with immobilized antibody, according to the man-
ufacturer’s protocol. Aliquots (20 ␮l) were subjected to SDS-PAGE,
except for omission of sample boiling, according to Fairbanks et al. (39)
or Laemmli (40), followed by Western blot analysis or autoradiography,
as appropriate.
Western Blot Analysis—For analysis of subcellular fractions, 30 ␮g of
protein were subjected to standard SDS-PAGE (40). In NaCl solubili-
zation experiments, the synaptic vesicle fraction (50 ␮g of protein) was
stirred in 0.2 ml of buffer containing 0.32 M sucrose, 4 mM Tris-maleate
(pH 7.4), and various concentrations of NaCl for 5 min at 4 °C and then
centrifuged at 200,000 ⫻ gmax for 1 h. The pellet was dissolved in 30 ␮l
of SDS sample buffer. The protein in the supernatant (180 ␮l) was
5931
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FIG. 3. Dependence of vesicular Glu uptake on the GAPDH/3-
PGK substrate. A, time course of vesicular Glu uptake induced by
activation of vesicle-bound GAPDH and 3-PGK. Glu uptake into syn-
aptic vesicles was measured at 30 °C for the indicated periods in the
absence (‚) or presence (●) of 2 mM GAP, 2 mM NAD, 2 mM Pi, and 0.1
mM ADP. B, GAPDH and 3-PGK substrate requirement for vesicular
Glu uptake. Glu uptake into synaptic vesicles was measured at 30 °C
after a 10-min incubation in the absence or presence of various agents
or exogenous 3-PGK (1 unit) as indicated. C, effect of various ADP
concentrations on Glu uptake into synaptic vesicles induced by activa-
tion of vesicle-bound GAPDH and 3-PGK. Glu uptake was determined
at 30 °C after 10-min incubation in the presence of 2 mM GAP, 2 mM
NAD, and 2 mM Pi. Values are the mean ⫾ S.D. of four separate
experiments.
precipitated with 15% trichloroacetic acid and dissolved in 30 ␮l of SDS
sample buffer. Aliquots (25 ␮l) of the 200,000 ⫻ gmax pellet and super-
natant fractions were subjected to standard SDS-PAGE.
For analysis of GAPDH and 3-PGK binding to synaptic vesicles,
synaptic vesicles (1 mg of protein) were washed twice by 20 ml of buffer
containing 0.32 M sucrose, 4 mM Tris-maleate (pH 7.4), and 0.8 M NaCl;
bound NaCl was then removed by washing twice with 20 ml of the same
buffer without NaCl. The washed synaptic vesicles (40 ␮g of protein)
5932 GAPDH and Vesicular Glu Accumulation

FIG. 4. Effect of various GAP and

exogenous ATP concentrations on
vesicular Glu uptake and ATP levels
after incubation. Glu uptake into syn-
aptic vesicles (A and C) and ATP levels in
the synaptic vesicle suspension (B and D),
after 10-min incubation at 30 °C, were
measured in the presence of 2 mM NAD, 2
mM Pi, 0.1 mM ADP, and the indicated
concentrations of GAP (A and B) or of

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various concentrations of exogenous ATP
(C and D). E, Glu uptake into synaptic
vesicles measured in the presence of var-
ious concentrations of GAP, 2 mM NAD, 2
mM Pi, and 0.1 mM ADP (●) or various
concentrations of exogenous ATP (‚) was
plotted as a function of ATP level in the
synaptic vesicle suspension, each deter-
mined after a 10-min incubation at 30 °C.
Values are the mean ⫾ S.D. of four sepa-
rate experiments.

were incubated at 37 °C for 10 min in 0.1 ml of buffer in the absence or
presence of 2 ␮g of purified GAPDH or 3-PGK. The synaptic vesicles
were pelleted by centrifugation at 200,000 ⫻ gmax for 1 h at 4 °C and
washed twice with the buffer. The pellet was suspended in 80 ␮l of SDS
sample buffer, and an aliquot (25 ␮l) was subjected to standard
SDS-PAGE.
Proteins were electrotransferred onto the Immobilon-P polyvinyli-
dene difluoride membrane (Millipore Corp.) using a semidry transfer
apparatus (Bio-Rad Trans-Blot SD). The membrane was treated with
5% nonfat dry milk in a solution containing 50 mM Tris-HCl (pH 7.4),
0.5 M NaCl, and 0.1% Tween 20 (TBS-T) for 1 h and then incubated for
2 h at room temperature with anti-GAPDH monoclonal antibody at a
1:50 dilution or anti-3-PGK polyclonal antibodies at 1:500 dilution,
followed by incubation with alkaline phosphatase-conjugated goat anti-
mouse IgG or anti-rabbit IgG, respectively, at room temperature for
1.5 h. Unbound antibodies were washed out with TBS-T. 5-Bromo-4-
chloro-3-indolyl phosphate and nitro blue tetrazolium (Bio-Rad) were
used as substrates for color development and analyzed using an image
analyzer (Bio-Rad Gel Doc 2000).
Glu Uptake into Synaptic Vesicles—Vesicular glutamate uptake was
measured by the filtration-based assay using Whatman GF/C filters, as
described previously (27, 28), with minor modifications. In the standard
assay, aliquots (10 ␮g of protein) of bovine synaptic vesicles were
incubated at 30 °C for 10 min with 100 ␮M [3H]Glu (a specific activity of
7.4 GBq/mmol was obtained by the addition of unlabeled Glu to
[3H]Glu) in 0.1 ml of an incubation medium (pH 7.4) containing 20 mM
Hepes-KOH, 0.25 M sucrose, 4 mM MgSO4, 4 mM KCl, and 2 mM
L-aspartic acid in the absence or presence of 2 mM ATP (pH adjusted to
7.4 by the addition of Tris-base). Prior to incubation, preincubation
without ATP and [3H]Glu was carried out for 1 min at 30 °C. When
inhibitor effect was tested, test agents were added at the start of the
preincubation period. In some experiments, ATP was replaced with a
mixture of 2 mM GAP, 2 mM Pi, 2 mM NAD, and 0.1 mM ADP (pH
adjusted to 7.4 by the addition of Tris-base) as a source of a Glu uptake
activator.
Glu Content in Synaptic Vesicles within Synaptosomes—[3H]Glu con-
tent in synaptic vesicles within the synaptosome was assayed as de-
scribed previously (42, 43) with minor modifications. Synaptosomes
(100 ␮g of protein) were suspended in 0.1 ml of oxygenated (95% O2, 5%
CO2) Krebs-Ringer buffer containing 150 mM NaCl, 2.4 mM KCl, 1.2 mM
Na2HPO4, 1.2 mM CaCl2, 1.2 mM MgSO4, 5 mM Hepes-Tris (pH 7.4), and
10 mM glucose and preincubated at 37 °C for 10 min in the absence or
presence of iodoacetate, oligomycin (Calbiochem), and pyruvate at in-
dicated concentrations. After 3 ␮Ci of [3H]Glu (42.0 Ci/mmol) were
added to the medium, synaptosomes were incubated for an additional
10 min. Aliquots (10 ␮l) were removed and filtered on Whatman GF/C
 GAPDH and Vesicular Glu Accumulation 5933
filters to determine the total amount of [3H]Glu taken up by the syn-
aptosomes, and the rest were immediately frozen on dry ice. For vesic-
ular [3H]Glu content determination, the frozen synaptosomes (90 ␮l)
were thawed by adding 1.5 ml of ice-cold hypotonic solution containing
6 mM Tris-maleate (pH 8.1) and 2 mM aspartate and incubated for 20
min at 0 °C. Aliquots (1 ml) were filtered on Whatman GF/C filters, and
radioactivity retained on filters was determined in a Beckman LS 6500
scintillation spectrophotometer.
Glu Release from Synaptosomes—Glutamate release from synapto-
somes was assayed using the superfusion technique described previ-
ously (43), with minor modifications. Synaptosomes (200 ␮g of protein)
were suspended in 1.5 ml of oxygenated (95% O2/5% CO2) artificial
cerebrospinal fluid (ACSF) containing 124 mM NaCl, 5 mM KCl, 2 mM
MgSO4, 1.25 mM NaH2PO4, 22 mM NaHCO3, and 10 mM D-glucose and
preincubated with or without 300 ␮M iodoacetate or 2 ␮M oligomycin at
37 °C for 10 min. After 3.5 ␮Ci of [3H]Glu (42.0 Ci/mmol) were added to
the medium, synaptosomes were incubated for an additional 10 min. An
aliquot (1.0 ml) of [3H]Glu-loaded synaptosomal suspension was layered
onto a cellulose-acetate membrane filter (pore size 0.45 ␮m) placed in a
superfusion chamber. The synaptosomes were superfused (0.5 ml/min)
with ACSF for 60 min before application of 50 ␮M 4-aminopyridine
(4-AP) plus 2 mM CaCl2 to trigger depolarization of the synaptosomal
membrane. In some control experiments, synaptosomes were super-
fused with ACSF containing 300 ␮M iodoacetate or 2 ␮M oligomycin for
the first 20 min of the superfusion period. This period was the same as
the sum of the synaptosomal preincubation and [3H]Glu-loading peri-

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ods. These procedures were all carried out at 37 °C. Fractions were
collected every 10 s for 3 min, from 30 s prior to 4-AP application. The
amount of [3H]Glu released into each fraction was expressed as a
percentage of the total [3H]Glu taken up into synaptosomes at the end
of 10 min of [3H]Glu loading. The total [3H]Glu loaded into synapto-
somes was calculated from the amount of [3H]Glu in 0.1 ml of loaded
synaptosomes, determined by the same filtration method used for the
vesicular Glu uptake assay.
Other Biochemical Analyses—GAPDH activity was measured using
20 ␮g of protein of synaptic vesicles in a reaction mixture (1 ml)
containing 0.1 M Tris-HCl (pH 8.5), 1.7 mM sodium arsenate, 20 mM
sodium fluoride, 1 mM NAD, 1 mM GAP, and 5 mM KH2PO4 (44).
Activity was calculated from the rate of increase in NADH formation by
monitoring absorbance at 340 nm at 25 °C.
ATP levels in the synaptic vesicle and synaptosome suspensions were
determined under the same conditions described for vesicular Glu up-
take and vesicular Glu content assays, respectively, by the luciferin/
luciferase method, using an ATP bioluminescent assay kit (Sigma-
Aldrich), according to the manufacturer’s protocol.
Generation of the membrane potential across the synaptic vesicle
membrane was monitored by ATP-induced fluorescence quenching of
the membrane potential-sensitive dye oxonol V (Molecular Probes, Inc.,
Eugene, OR) using a Fluorolog III fluorospectrophotometer (Horiba
Jobin Yvon Co., Ltd., Tokyo, Japan) as described previously (32, 43).
Vesicular ATPase activity was assayed by determining free inorganic
phosphate liberated upon incubation of synaptic vesicles (30 ␮g of
protein) with ATP, according to the method of Lanzetta et al. (45), with
minor modifications as described previously (43).
RESULTS
A 37-kDa Protein Enriched in Synaptic Vesicles Is Labeled
with [3-32P]1,3-BPG and Identified as GAPDH—Purified syn-
aptic vesicles were allowed to react with [3-32P]1,3-BPG for 10 s
and subjected to SDS-PAGE at either neutral (Fig. 1A) or
alkaline (Fig. 1B) pH (39, 40). Electrophoresis at neutral pH
revealed incorporation of a radioactive moiety of [3-32P]1,3-
BPG into vesicular proteins of Mr ⫽ 37,000 and 29,000. In
contrast, electrophoresis at alkaline pH revealed only the 29-
kDa protein, which appears to retain more of the 32P label than
when electrophoresis was conducted at neutral pH. These re-
sults indicate that both the 29- and 37-kDa vesicular proteins
can incorporate a 32P-containing moiety; however, the stability
of the labeled proteins differs depending on pH. Incorporation
of the radioactive moiety into the 37-kDa protein was stimu-
lated by Mg2⫹ but not affected by 3-PG. Labeling of the 29-kDa
protein was inhibited by Mg2⫹ and completely blocked by 3-PG.
The linkage of the 32P-containing moiety to the 37-kDa protein
was labile in the presence of 0.2 M hydroxylamine (data not
shown). These observations suggested that the 37- and 29-kDa
FIG. 5. Iodoacetate selectively inhibits GAPDH/3-PGK activa-
tion-dependent over exogenous ATP-dependent vesicular Glu
uptake. A, effect of various iodoacetate concentrations on GAPDH/3-
PGK activation-dependent versus exogenous ATP-dependent vesicular
Glu uptake. The synaptic vesicle fraction (10 ␮g of protein) was prein-
cubated at 30 °C for 10 min in the presence of various concentrations of
iodoacetate, followed by an additional 10-min incubation at 30 °C after
the addition of a mixture of [3H]Glu, 2 mM GAP, 2 mM NAD, 2 mM Pi,
and 0.1 mM ADP (●) or of a mixture of [3H]Glu and 2 mM ATP (‚).
[3H]Glu accumulated into synaptic vesicles was determined as de-
scribed under “Experimental Procedures.” B, effect of various iodoac-
etate concentrations on vesicle-bound GAPDH activity and on ATP
produced by activation of vesicle-bound GAPDH/3-PGK. The synaptic
vesicle fraction (10 ␮g of protein) was incubated in the presence of
various concentrations of iodoacetate for a total period of 20 min as
described for A, except for replacement of [3H]Glu with nonradioactive
Glu. After incubation, GAPDH activity (●) and the amount of ATP
produced (䡺) were determined, as described under “Experimental Pro-
cedures.” Values are the mean ⫾ S.D. of four separate experiments.
proteins might be GAPDH and PGM, respectively. This was
confirmed by immunoprecipitation with antibodies directed
against these proteins (Fig. 1C). Thus, labeling of the 37-kDa
GAPDH with [3-32P]1,3-BPG probably occurs by formation of a
thioester bond between a cysteine residue and the 3-phospho-
glyceroyl moiety of 1,3-BPG; thioester bonds are known to be
labile at alkaline pH. Labeling of the 29-kDa PGM probably
represents phosphorylation of a histidine residue that is labile
at either neutral or acidic pH.
In order to determine the subcellular distribution of GAPDH
and PGM, we examined the amount of [3-32P]1,3-BPG radioac-
tivity incorporated into these two proteins present in synapto-
somal cytosol, perikaryal cytosol, microsome, synaptic vesicle,
and plasma membrane fractions (Fig. 1D). For each subcellular
fraction, an equivalent amount of protein was incubated with
[3-32P]1,3-BPG. Labeled GAPDH was found in all subcellular
5934 GAPDH and Vesicular Glu Accumulation
FIG. 6. Comparison in responsive-
ness to chloride, aspartate, t-ACPD,
Rose Bengal, and FCCP between
GAPDH/3-PGK activation-dependent
and exogenous ATP-dependent vesic-
ular Glu uptake. The effect of various
chloride concentrations on Glu uptake
into synaptic vesicles was determined at
30 °C after a 10-min incubation in the
presence of 2 mM GAP, 2 mM NAD, 2 mM
Pi, and 0.1 mM ADP (A) or of 2 mM ATP
(B). Effect of 2 mM Asp, 2 mM t-ACPD, 0.5
␮M Rose Bengal (RB), and 25 ␮M FCCP on
Glu uptake into synaptic vesicles was de-
termined at 30 °C after 10-min incubation
in the presence of 2 mM GAP, 2 mM NAD,
2 mM Pi, and 0.1 mM ADP (C) or of 2 mM
ATP (D). Values are the mean ⫾ S.D. of
four separate experiments.
fractions but was enriched in the synaptic vesicle fraction. In
contrast, PGM was most enriched in the synaptosomal cytosol.
Subcellular fractions were also subjected to Western blotting
with anti-GAPDH antibody. GAPDH was found in the greatest
concentration in the synaptic vesicle fraction (Fig. 1E), in ac-
cord with the results obtained with the labeling method. Anal-
ysis of subcellular fractions indicates that GAPDH not only
occurs in the cytosol but also can bind to various types of
membranes. To determine the nature of the interaction of
GAPDH with synaptic vesicles, synaptic vesicles were treated
with various concentrations of NaCl (Fig. 1F). GAPDH was
dissociated from synaptic vesicles with increasing salt concen-
trations. At 0.8 M NaCl, only about 10% of GAPDH was found
to remain associated with synaptic vesicle membranes. At
physiologic ionic strength, GAPDH is associated with synaptic
vesicle membranes, although it does also occur in the cytosol
fractions.
3-PGK Is Associated with Synaptic Vesicles via Interaction
with GAPDH—In the glycolytic pathway, GAPDH is known to
exist in a complex with 3-PGK (46). The activities of GAPDH
and 3-PGK are energetically coupled, utilizing GAP, NAD, Pi,
and ADP, to yield 3-PG, ATP, NADH, and H⫹ (46, 47). Because
GAPDH is enriched in synaptic vesicles, we considered the
possibility that 3-PGK may also preferentially localize to syn-
aptic vesicle membranes. Western blots were conducted on
subcellular fractions using anti-3-PGK antibody (Fig. 2A). Like
GAPDH, 3-PGK was found enriched in the synaptic vesicle
fraction. Immunoreactive 3-PGK was also dissociated from syn-
aptic vesicles by increasing concentrations of NaCl in a manner
similar to that observed for GAPDH (Fig. 2B). To determine
whether GAPDH and 3-PGK exist in a complex on synaptic
vesicles, we performed experiments to determine whether
GAPDH and 3-PGK could be co-immunoprecipitated. Synaptic
vesicle salt extracts were incubated with anti-3-PGK antibody,
and the resulting immunoprecipitates were subjected to West-
ern blotting with anti-GAPDH antibody (Fig. 2C). Immunore-
active GAPDH was detected in the anti-3-PGK antibody immu-
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noprecipitates, indicating that GAPDH and 3-PGK must exist
in a tight complex. A second co-immunoprecipitation experi-
ment was carried out, which showed that 3-PGK can be co-
precipitated with anti-GAPDH antibody (Fig. 2D). In order to
determine whether GAPDH and 3-PGK directly bind to the
synaptic vesicles, synaptic vesicles were first washed with 0.8 M
NaCl and then incubated with purified GAPDH and/or 3-PGK.
The vesicles were pelleted by centrifugation, and the amount of
vesicle-bound GAPDH or 3-PGK was determined by immuno-
blotting (Fig. 2E). GAPDH was found capable of binding to the
synaptic vesicle with or without the addition of 3-PGK. In
contrast, binding of 3-PGK to synaptic vesicles was greatly
increased when GAPDH was included with 3-PGK. These re-
sults suggest that GAPDH directly binds to synaptic vesicles
and that 3-PGK binds to vesicle-bound GAPDH.
Synaptic Vesicles Are Capable of Accumulating Glu via Ac-
tivation of Endogenous GAPDH/3-PGK—Synaptic vesicles
bearing both GAPDH and 3-PGK should be able to synthesize
ATP when GAPDH substrates and ADP are supplied. GAPDH
and 3-PGK activity could regenerate ATP for vesicular uptake
of Glu. Synaptic vesicles were incubated for various periods
with [3H]Glu in the presence or absence of 2 mM GAP, NAD,
and Pi and 0.1 mM ADP. Vesicular Glu uptake was dependent
on the presence of the GAPDH/3-PGK substrate mixture
throughout the incubation period(s) tested and was approxi-
mately linear up to 6 min under these conditions (Fig. 3A). When
any one of the components was omitted from the incubation
mixture, vesicular Glu uptake was not supported (Fig. 3B).
[3H]Glu uptake resulting from ATP generated by vesicular
GAPDH and 3-PGK was comparable with that observed with 2
mM ATP and not increased by the addition of exogenous 3-PGK.
ADP supported uptake of [3H]Glu in a concentration-dependent
manner but was inhibitory at concentrations above 0.1 mM (Fig.
3C). These results indicate that vesicular Glu uptake can occur
by harnessing the activity of vesicle-bound GAPDH and 3-PGK.
ATP Synthesized by Vesicle-bound GAPDH/3-PGK Is More
Effective than Exogenous ATP in Glu Uptake—Experiments
 GAPDH and Vesicular Glu Accumulation
FIG. 7. Effect of GAPDH substrates on vesicular Glu uptake in
the presence of ATP. Glu uptake into isolated synaptic vesicles at
30 °C after 10 min incubation in the presence of 30 ␮M ATP (A) or 2 mM
ATP (B) was measured in the absence or presence of GAPDH substrates
(2 mM GAP, 2 mM NAD, and 2 mM Pi) as described under “Experimental
Procedures.” Values are the mean ⫾ S.D. of four separate experiments.
shown in Fig. 3 suggested that GAP-mediated vesicular Glu
uptake probably occurs as a result of ATP synthesis. To directly
demonstrate this, synaptic vesicles were incubated with vari-
ous concentrations of GAP in the presence of 2 mM NAD, Pi, and
0.1 mM ADP, and the amount of ATP produced, as well as Glu
uptake, was determined (Fig. 4, A and B). ATP was synthesized
in a GAP-concentration-dependent manner in parallel to vesic-
ular Glu uptake. Under these conditions, 12 ␮M ATP were
sufficient to give rise to maximal vesicular Glu uptake. This
concentration is substantially lower than the apparent Km
value for ATP (0.8 –1.2 mM), determined using exogenous ATP
under standard assay conditions for vesicular Glu uptake (28)
(see Fig. 9B). These observations raised the possibility that
ATP produced by vesicle-bound GAPDH/3-PGK might be more
effective than exogenous ATP in supporting vesicular Glu up-
take. To further explore this possibility, exogenous ATP-de-
pendent vesicular Glu uptake was compared with GAP-sup-
ported Glu uptake. Synaptic vesicles were incubated with
various concentrations of exogenous ATP, and the amount of
ATP, as well as Glu uptake, after 10 min of incubation was
determined (Fig. 4, C and D). At least 80 –100 ␮M exogenous
ATP were needed to support the same maximal Glu uptake
observed for GAP-supported uptake. This ATP concentration is
much higher than that observed with the endogenous ATP-
generating system. In Fig. 4E, the amount of Glu taken up into
synaptic vesicles was plotted as a function of the ATP concen-
tration determined after a 10-min incubation. The amount of
5935
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FIG. 8. ATP-dependent vesicular Glu uptake is inhibited by
ADP, which has no effect on vesicular ATPase activity. A, effect
of various concentrations of ADP (●), AMP (䡺), or adenosine (‚) on Glu
uptake into synaptic vesicles at 30 °C after 10 min incubation was
determined in the presence of 2 mM ATP. B, effect of various ADP
concentrations on vesicular ATPase activity was determined as de-
scribed under “Experimental Procedures.” Values are the mean ⫾ S.D.
of four separate experiments.
Glu accumulated per ATP level after 10 min of incubation is
greater when GAPDH/3-PGK substrates are utilized. These
results suggest that the ATP generated by vesicle-bound
GAPDH/3-PGK is more effective than exogenous ATP in sup-
porting vesicular Glu uptake.
GAP-dependent Vesicular Glu Uptake Is Inhibited by Iodoac-
etate—In an effort to provide further evidence that GAP-de-
pendent Glu uptake involves GAPDH, we have determined the
effect of the GAPDH-selective inhibitor iodoacetate on vesicu-
lar Glu uptake in the presence of GAP, NAD, Pi, and ADP. As
shown in Fig. 5A, iodoacetate inhibited vesicular GAP-depend-
ent Glu uptake in a concentration-dependent manner. When
ATP was substituted for the GAPDH/3-PGK substrates, iodoac-
etate exhibited little inhibition in the concentration range
tested. Iodoacetate also inhibited GAPDH activity and im-
paired the ability of synaptic vesicles to regenerate ATP from
ADP in a concentration-dependent manner. These results in-
dicate that iodoacetate prevents, via inhibition of GAPDH, ATP
generation necessary for vesicular Glu uptake.
Characterization of the GAPDH/3-PGK Activation-dependent
Vesicular Glu Uptake System—The ATP-dependent vesicular
Glu uptake system is known to be markedly stimulated by
low millimolar chloride (28, 34, 48 –50) and harnesses an
electrochemical proton gradient as the driving force (28, 29 –
34). In contrast to Na⫹-dependent plasma membrane Glu
uptake, vesicular Glu uptake is insensitive to aspartate (28,
5936 GAPDH and Vesicular Glu Accumulation
FIG. 9. Kinetics of ADP inhibition of vesicular Glu uptake. A,
the initial rate of vesicular Glu uptake at 30 °C in the presence of 2 mM
ATP was determined with various Glu concentrations, each in the
absence (E) or presence of 0.5 mM (Œ) or 2 mM ADP (f), by incubating
the synaptic vesicle fraction (10 ␮g of protein) for 1.5 min, as described
under “Experimental Procedures.” B, the initial rate of vesicular Glu
uptake with a fixed Glu concentration (100 ␮M) was determined with
various ATP concentrations in the absence (E) or presence of 0.5 mM (Œ)
or 2 mM ADP (f), as described above. Values are the mean ⫾ S.D. of four
separate experiments.
31, 34, 48, 50) but potently inhibited by the membrane-
permeant polyhalogenated fluorescein Rose Bengal (43). t-
ACPD also inhibits vesicular Glu uptake by serving as a
substrate for the vesicular Glu transporter (51).
We have studied the characteristics of GAPDH/3-PGK acti-
vation-induced vesicular Glu uptake with respect to the prop-
erties of the ATP-dependent vesicular Glu uptake system.
When vesicle-bound GAPDH and 3-PGK were activated in the
presence of their substrates and various concentrations of chlo-
ride, maximal Glu uptake occurred at 4 mM chloride; at higher
chloride concentration, Glu uptake was attenuated (Fig. 6A).
This dependence on chloride is indistinguishable from that
observed for exogenous ATP-induced vesicular Glu uptake (Fig.
6B). Moreover, GAPDH/3-PGK activation-induced vesicular
Glu uptake was insensitive to Asp but affected by t-ACPD and
Rose Bengal as well as by the proton ionophore FCCP, in a
manner similar to that observed with exogenous ATP-depend-
ent Glu uptake (Fig. 6, C and D). These results indicate that
the Glu transporter responsible for GAPDH/3-PGK activation-
induced vesicular Glu uptake is the same as that responsible
for ATP-dependent vesicular Glu uptake.
GAPDH Substrates Enhance Vesicular Glu Uptake in the
Presence of a Low Concentration of ATP—Since ATP generated
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FIG. 10. Effect of ADP on membrane potential in synaptic ves-
icles. After oxonol V (1.3 ␮M) was allowed to equilibrate with synaptic
vesicles as described under “Experimental Procedures,” ATP was added
(2 mM as a final concentration). Various amounts of ADP were then
added, resulting in indicated final concentrations, followed by the ad-
dition of FCCP (8.33 ␮M as a final concentration). A, time course tracing
of fluorescence quenching before and after the addition of 0.3, 1, and 2
mM ADP. The data shown are representative of three separate experi-
ments. B, membrane potential as a function of various concentrations
of ADP as indicated. Values are the mean ⫾ S.D. of three separate
experiments.
by vesicle-bound GAPDH/3-PGK appeared more effective than
exogenous ATP in supporting vesicular Glu uptake, it was of
interest to see whether GAPDH substrates are effective in
augmenting vesicular Glu uptake in the presence of limiting
concentrations of ATP. Fig. 7 shows the effect of GAPDH sub-
strates on vesicular Glu uptake in the presence of ATP. The
addition of a mixture of GAP, NAD, and Pi, which alone exhib-
ited no Glu uptake, elevated Glu uptake in the presence of a
low concentration of exogenous ATP (Fig. 7A). This suggests
that ADP generated by ATPase is recycled to ATP by 3-PGK,
when vesicle-bound GAPDH is activated, and that the regen-
erated ATP is utilized to give rise to additional Glu uptake. At
a much higher ATP concentration, this augmentation was
much smaller, however (Fig. 7B). This could be partly attrib-
uted to saturation of proton pump ATPase with ATP. These
results are compatible with the notion that GAPDH/3-PGK-
 GAPDH and Vesicular Glu Accumulation
FIG. 11. Iodoacetate reduces vesicular [3H]Glu content in syn-
aptosomes more effectively than oligomycin, whereas both
agents have similar effects on synaptosomal ATP levels in the
concentration ranges tested. Synaptosomes were preincubated at
37 °C for 10 min in the presence of various concentrations of iodoacetate
(A, C, E, and G) or oligomycin (B, D, F, and H), followed by the addition
of [3H]Glu; ATP level (A and B), synaptosomal [3H]Glu uptake (C and
D), and vesicular [3H]Glu content (E and F) were determined at 37 °C
after 10 min, as described under “Experimental Procedures.” Vesicular
[3H]Glu content was expressed as percentage of total synaptosomal
[3H]Glu uptake (G and H). Values are the mean ⫾ S.D. of four separate
experiments.
mediated vesicular Glu uptake occurs through the ATP-de-
pendent vesicular Glu uptake system.
ADP Inhibits ATP-dependent Vesicular Glu Uptake—In or-
der to understand the ADP inhibition observed earlier (Fig.
3C), we have studied the effect of ADP on vesicular Glu uptake
supported by exogenous ATP. It was found that exogenous
ATP-dependent vesicular Glu uptake was also inhibited by
ADP, but not by AMP or adenosine, at concentrations higher
than 0.1 mM (Fig. 8A). ADP had no effect on ATPase activity at
the concentrations tested up to 10 mM (Fig. 8B). Kinetic exper-
iments indicate that this inhibition is noncompetitive with
respect to Glu or ATP (Fig. 9, A and B). Thus, neither Km for
Glu nor Km for ATP was altered significantly; Km values for Glu
5937
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FIG. 12. Effect of pyruvate, glucose, oligomycin, and iodoac-
etate on vesicular [3H]Glu content and ATP levels in synapto-
somes. Synaptosomes were preincubated at 37 °C for 10 min in the
absence or presence of 10 mM glucose, 2 ␮M oligomycin, 300 ␮M iodoac-
etate, or 1 mM pyruvate; ATP level (A) and vesicular [3H]Glu content (B)
were determined at 37 °C after an additional 10-min incubation with
[3H]Glu, as described in the legend to Fig. 11. Vesicular [3H]Glu content
was expressed as percentage of total synaptosomal [3H]Glu uptake.
Values are the mean ⫾ S.D. of four separate experiments.
in the presence of 0, 0.5, and 2.0 mM ADP were 1.6, 1.6, and 1.5
mM, respectively, and those for ATP in the presence of 0, 0.5,
and 2.0 mM ADP were 0.82, 0.76, and 0.79 mM, respectively.
The noncompetitive inhibition with respect to ATP was rather
surprising but was consistent with ADP’s inability to inhibit
ATPase activity (Fig. 8B).
The effect of ADP on membrane potential was examined in
an effort to gain further insight into the mechanism of ADP
inhibition. Fig. 10A shows that ADP is capable of dissipating
preformed membrane potential in a concentration-dependent
manner. Disruption of membrane potential by 10 mM ADP was
47% (Fig. 10B). ADP did not completely dissipate membrane
potential at the concentrations tested. The ability of ADP to
reduce membrane potential would not entirely account for its
inhibitory effect on Glu uptake.
ATP Produced by Glycolysis Is Predominantly Utilized for
Vesicular Accumulation of Glu in the Nerve Ending—The data
indicating that ATP produced by vesicular GAPDH and 3-PGK
can support vesicular uptake of Glu prompted us to postulate
that Glu accumulation into synaptic vesicles in the nerve ter-
5938 GAPDH and Vesicular Glu Accumulation

FIG. 14. Proposed role of GAPDH and 3-PGK in vesicular Glu

FIG. 13. Treatment of synaptosomes with iodoacetate prior to
and during the [3H]Glu-loading period reduces [3H]Glu release.
Synaptosomes were preincubated at 37 °C for 10 min in the absence (E,
●, 䡺, and ‚) or presence of 300 ␮M iodoacetate (IA; f) or 2 ␮M oligo-
mycin (OM; Œ); [3H]Glu was loaded into the synaptosome at 37 °C for 10
min. The [3H]Glu-loaded synaptosomes were layered onto the mem-
brane in the superfusion chamber and superfused at 37 °C for 20 min
uptake. The glycolytic intermediate GAP is converted to 1,3-BPG by
synaptic vesicle-associated GAPDH in the presence of NAD and Pi.
1,3-BPG is then converted to 3-PG by GAPDH-associated 3-PGK, ac-
companied by phosphorylation of ADP to form ATP. ATP thus produced
is efficiently consumed by vesicular H⫹-ATPase, generating an electro-
chemical proton gradient in the presence of low concentrations of chlo-
ride; this provides the driving force for Glu uptake, which is carried out
by the vesicular Glu transporter (VGLUT). ADP generated by vesicular
H⫹-ATPase is efficiently converted to ATP by 3-PGK. When ADP is
accumulated, Glu uptake into synaptic vesicles would be inhibited. ⌬⌿,
membrane potential; ⌬pH, pH gradient.

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with ACSF in the absence (E, ●, f, and Œ) or presence of 300 ␮M
iodoacetate (䡺) or 2 ␮M oligomycin (‚). After further superfusion with mitochondria. However, pyruvate failed not only to enhance
ACSF at 37 °C for 40 min, synaptosomes were subjected to depolariza- vesicular Glu accumulation in the absence of exogenous glucose
tion with 50 ␮M 4-aminopyridine (4-AP; ●, f, Œ, 䡺, ‚) or not (E); but also to overcome the reduction of vesicular Glu content
[3H]Glu release was measured as described under “Experimental Pro-
cedures.” Values are the mean ⫾ S.D. of six separate experiments.
caused by iodoacetate (Fig. 12B). Oligomycin blocked pyruvate-
induced increases in ATP levels (Fig. 12A) but had no signifi-
cant effect on vesicular Glu content corrected for synaptosomal
minal may be supported by glycolytically produced ATP. In
uptake (Fig. 12B). In the presence of glucose, pyruvate failed to
order to test this hypothesis, we compared the effects of the
elevate ATP levels or vesicular Glu content, either in the ab-
GAPDH inhibitor iodoacetate and the mitochondrial ATP syn-
sence or presence of oligomycin. These results indicate that
thesis inhibitor oligomycin on vesicular accumulation of Glu in
ATP produced by glycolysis is predominantly utilized, whereas
purified synaptosomes. When GAPDH was inhibited by iodoac-
mitochondria-generated ATP is insufficient to support vesicu-
etate, or mitochondrial ATP synthesis was inhibited by oligo-
lar uptake of Glu in nerve endings. These observations support
mycin, ATP levels were reduced in a concentration-dependent
the notion that synaptic vesicle-bound GAPDH and 3-PGK play
manner (Fig. 11, A and B). The reduction in synaptosomal ATP
an important role by providing ATP necessary for Glu uptake
caused by 500 ␮M iodoacetate and 2 ␮M oligomycin was 67 and
into synaptic vesicles in the nerve terminal.
64%, respectively. Both reagents decreased synaptosomal
Glu Accumulated into Vesicles, at the Expense of Glycolyti-
[3H]Glu uptake to a similar extent (Fig. 11, C and D). Inhibition
cally Produced ATP, Is Released upon Depolarization—In order
of synaptosomal [3H]Glu uptake by 500 ␮M iodoacetate and 2
to determine whether the Glu accumulated into synaptic vesi-
␮M oligomycin was 33 and 37%, respectively. In contrast, io-
cles by glycolysis-generated ATP is released upon nerve ending
doacetate was more effective than oligomycin in reducing ve-
stimulation, we examined depolarization-induced release in
sicular [3H]Glu content (Fig. 11, E and F). When vesicular
synaptosomes. Synaptosomes treated with either iodoacetate
content was corrected for synaptosomal uptake (Fig. 11, G and
or oligomycin, prior to (and during) loading with [3H]Glu, were
H), the inhibition of vesicular uptake within the synaptosomes
depolarized by the potassium channel blocker 4-AP. As shown
by 500 ␮M iodoacetate was 78%, whereas the inhibition by 2 ␮M
in Fig. 13, iodoacetate treatment led to substantial reduction in
oligomycin was only 16%. Although the reduction of [3H]Glu
the amount of [3H]Glu released. In contrast, oligomycin pre-
content by iodoacetate is probably due to inhibition of GAPDH,
treatment had a much smaller effect. The reduction in depo-
the possibility is not entirely ruled out that iodoacetate could
larization-induced Glu release is similar to the reduction of
inhibit vesicular Glu uptake within the synaptosomes by a
vesicular Glu content observed with each inhibition (Fig. 11, G
mechanism not involving GAPDH inhibition. However, iodoac-
and H). When synaptosomes were treated with iodoacetate or
etate did not inhibit [3H]Glu uptake by purified synaptic vesi-
oligomycin after loading with [3H]Glu, release was affected
cles in the presence of ATP (Fig. 5A).
only to a small extent. The magnitude of this reduction was
Inhibition of GAPDH by iodoacetate would lead to reduction
similar to that observed when synaptosomes were treated with
of pyruvate, the precursor for the tricarboxylic acid cycle sub-
oligomycin before loading with [3H]Glu.
strate acetyl-CoA and thereby would decrease ATP production
The large differential of [3H]Glu release observed between
in mitochondria. To better understand the mechanism of vesic-
iodoacetate- and oligomycin-treated synaptosomes indicates
ular Glu accumulation, we further investigated the effect of
that [3H]Glu accumulated in synaptic vesicles utilizing ATP
pyruvate on vesicular Glu uptake in synaptosomes in the ab-
synthesized via glycolysis can be released by depolarization.
sence or presence of iodoacetate or oligomycin. Pyruvate led to
Thus, the effect of GAPDH inhibition by iodoacetate on presyn-
an increase in ATP levels when added to synaptosomes incu-
aptic vesicular Glu accumulation is reflected in a decrease of
bated without glucose or to iodoacetate-treated synaptosomes
Glu released by exocytosis.
incubated with glucose (Fig. 12A). Increased levels of ATP
resulting from incubation with pyruvate were not observed in DISCUSSION
the presence of oligomycin. These results are consistent with We have presented evidence that GAPDH is modified by the
the concept that pyruvate-induced ATP production occurs in high energy glycolytic intermediate 1,3-BPG via covalent at-
 GAPDH and Vesicular Glu Accumulation
tachment of the 3-phosphoglyceroyl moiety. The radioactive
moiety was removed by treatment with neutral hydroxylamine.
This suggests that this modification involves thioester forma-
tion between the 3-phosphoglyceroyl group and a GAPDH cys-
teine residue, most likely representing the acylated enzyme
intermediate (52, 53).
GAPDH was found to be bound to purified synaptic vesicles.
This observation is in agreement with the findings by Schlaefer
et al. (54) and Rogalski-Wilk and Cohen (55). Schlaefer et al.
have postulated that vesicle-associated GAPDH might be func-
tionally coupled to ATP uptake into synaptic vesicles (54). We
have provided evidence that synaptic vesicle-bound GAPDH,
coupled with 3-PGK, can produce ATP sufficient for Glu uptake
by the vesicular Glu uptake system. In the presence of the
glycolytic intermediate GAP, GAPDH produces 1,3-BPG, which
is then converted to 3-PG and ATP by the GAPDH-bound
3-PGK. This conversion is coupled, since GAPDH and 3-PGK
exist in a complex (Fig. 2). Bernhard and co-workers (46, 47)
have demonstrated direct transfer of 1,3-BPG from GAPDH to
3-PGK in muscle tissue. Inhibition of vesicle-bound GAPDH
activity by iodoacetate led to a reduction of ATP synthesis as
well as to an attenuation of Glu uptake into synaptic vesicles
(Fig. 5). This was also observed in synaptosomes (Figs. 11 and
12). Iodoacetate decreased the amount of exocytotically re-
leased Glu (Fig. 13). Thus, it is likely that, in synaptosomes, the
ATP generated from the glycolytic intermediates provides the
energy required for the majority of the Glu taken up into
synaptic vesicles. Oligomycin inhibition of mitochondrial ATP
synthesis had little effect on vesicular Glu accumulation in
synaptosomes (Figs. 11 and 12). Moreover, substitution of
pyruvate for glucose, which increased ATP levels in synapto-
somes, failed to elevate vesicular Glu content (Fig. 12). These
observations indicate that mitochondrially produced ATP con-
tributes minimally to Glu transport into synaptic vesicles in
synaptosomes, suggesting the critical importance of glycolyti-
cally produced ATP in vesicular Glu accumulation in the pre-
synaptic nerve terminal.
In cardiac myocytes, there is evidence that glycolysis-derived
ATP is preferentially used to prevent ATP-sensitive K⫹ chan-
nels from opening, thereby maintaining cellular membrane
potential (56). In the skeletal muscle, evidence suggests that
fast twitch fibers of the white vastus utilize ATP produced by
glycolysis, whereas the slow twitch fibers of the soleus largely
use ATP synthesized by mitochondria. The former has a high
glycolytic capacity and a low oxidative capacity, in contrast to
the latter, which is opposite (57). GAPDH activity is 3– 4 times
higher in the white quadriceps than in the soleus (58). GAPDH
is a component associated with the skeletal muscle junctional
“foot,” which links the transverse tubule and terminal cister-
nae, forming the triad, the site of excitation-contraction cou-
pling (59, 60). Heilmeyer et al. (60) have demonstrated that the
junctional triad is capable of producing ATP from GAP, NAD,
Pi, and ADP, indicating that localized glycolytic ATP synthesis
can occur at the site where an energy supply is immediately
needed. These observations are consistent with the concept
that rapid, energy-consuming cellular processes rely on glyco-
lytically produced ATP.
ATP generated at the surface of the synaptic vesicle may
play an essential role in ensuring prompt Glu refilling of syn-
aptic vesicles in neurons. Mitochondria alone may not entirely
be able to meet the ATP requirement for maintaining normal
neurotransmission, an extremely rapid cellular process. Syn-
aptic vesicles release their contents and then must be rapidly
refilled in order to continuously support normal neurotrans-
mission (61). Energy demand for vesicular uptake would be
dynamic and reflect nerve activity. The synaptic vesicle’s func-
5939
tion of refilling Glu would be more efficiently served if ATP
were generated “locally” by vesicle-bound GAPDH/3-PGK. In
isolated synaptic vesicles, GAPDH/3-PGK-generated ATP ap-
pears more effective than exogenous ATP in supporting vesic-
ular Glu uptake (Fig. 4). In neurons, mitochondria located in
the nerve ending may not be close enough to synaptic vesicles
to provide sufficient ATP in a timely fashion for sustaining
adequate Glu accumulation. Moreover, ATP synthesis in mito-
chondria occurs after ATP formation during glycolysis. Thus,
the synaptic vesicle-bound GAPDH-3-PGK system would have
an advantage over mitochondrial ATP synthase in its prompt
response to energy demand for refilling vesicles with the trans-
mitter Glu.
In conclusion, we propose that the ATP produced by vesicle-
bound GAPDH and 3-PGK plays a crucial role in swiftly refill-
ing vesicles with the major excitatory neurotransmitter Glu
(and possibly other neurotransmitters) in the presynaptic
nerve terminal. According to our hypothesis depicted in Fig. 14,
GAP produced via glucose metabolism is converted to 1,3-BPG
at the expense of NAD and Pi by synaptic vesicle-bound
GAPDH; 1,3-BPG transfers its high energy phosphate to ADP,
catalyzed by GAPDH-bound 3-PGK, forming ATP at the sur-
face of vesicles. This ATP is utilized by vesicular H⫹-pump
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ATPase to generate an electrochemical proton gradient across
the vesicle membrane, providing the driving force for Glu
transport into synaptic vesicles. ADP produced by ATPase
would be rapidly recycled to ATP by the GAPDH-3PGK system
in the presence of glycolysis, eliminating its potential inhibi-
tory effect on Glu uptake. Acute depletion of glucose could lead
to a reduction of ATP produced via glycolysis within the micro-
environment of the presynaptic nerve terminal. This would
diminish Glu transport into the vesicle, resulting in a de-
creased amount of Glu released, thereby attenuating Glu-me-
diated synaptic transmission. Moreover, in the prolonged ab-
sence of glycolysis, excess ADP would be accumulated, im-
pairing the ATP-dependent vesicular uptake system, further
contributing to reduction in vesicular Glu content and release.
The mechanism outlined above could offer insight into our
molecular understanding of hypoglycemia-induced, abnormal
electrophysiological activity and resulting clinical symptoms as
well as into the essential requirement for glucose metabolism
in normal synaptic transmission.
Acknowledgments—We are grateful to Dr. Kiyokazu Ogita (Setsunan
University, Hirakata, Japan) for technical advice in the superfusion
release assay, Dr. Koji Hirata for synaptic vesicle preparation, and
Oriental Yeast Co., Ltd., Tokyo, for the kind gift of affinity-purified
polyclonal antibodies to recombinant PGM type B. We also thank Mary
Roth for excellent assistance in preparation of the manuscript.
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MEMBRANE TRANSPORT STRUCTURE
FUNCTION AND BIOGENESIS:
Glycolysis and Glutamate Accumulation
into Synaptic Vesicles: ROLE OF
GLYCERALDEHYDE PHOSPHATE
DEHYDROGENASE AND
3-PHOSPHOGLYCERATE KINASE

Atsushi Ikemoto, David G. Bole and

Tetsufumi Ueda
J. Biol. Chem. 2003, 278:5929-5940.
doi: 10.1074/jbc.M211617200 originally published online December 17, 2002

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