(Received for publication, September 16, 1994; and in revised form, November 18, 1994)
From the
The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and
GAD65, synthesize the neurotransmitter -aminobutyric acid in
neurons and pancreatic
-cells. Previous studies suggest that GAD67
is a soluble cytosolic protein, whereas GAD65 is membrane-associated.
Here, we study the intracellular distribution of GAD67 in neurons,
pancreatic
-cells, and fibroblasts transfected either with GAD65
and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially
recovered with GAD65 in membrane-containing pellet fractions and Triton
X-114 detergent phases. The two proteins co-immunoprecipitate from
extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not
when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and
used as a starting material for immunoprecipitation. GAD67 is
concentrated in the Golgi complex region in GAD65-GAD67 co-transfected
fibroblasts, but not in fibroblasts transfected with GAD67 alone. A
pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex
region and in many synapses. The two proteins also co-localize in the
perinuclear region of some pancreatic
-cells. GAD67 interacts with
the NH
-terminal region of GAD65, even in the absence of
palmitoylation of this region of GAD65. Taken together, our results
indicate that GAD65-GAD67 association occurs in vivo and is
required for the targeting of GAD67 to membranes.
Glutamic acid decarboxylase (GAD) ()synthesizes the
neurotransmitter
-aminobutyric acid (GABA) in neurons and
pancreatic
-cells. The enzyme exists as two isoforms of 67 kDa
(GAD67) (1) and 65 kDa (GAD65)(2) . Both isoforms
catalyze the conversion of L-glutamic acid into GABA, but
interact differently with the co-factor pyridoxal 5`-phosphate,
suggesting that their enzymatic activity is differently regulated in vivo(3, 4, 5) . GAD65 is a major
autoantigen in insulin-dependent diabetes mellitus (6, 7, 8) and stiff-man
syndrome(9, 10, 11) , a rare disease of the
central nervous system with which insulin-dependent diabetes mellitus
is frequently associated(12) .
GAD67 and GAD65 are highly
homologous (65% identity in humans) but have distinct biochemical
properties and intracellular distributions. Following subcellular
fractionation of rat pancreatic islets(13, 14) and
Chinese hamster ovary (CHO) cells transfected either with GAD65 or
GAD67(15) , GAD65 is partially recovered in the high speed
pellet, whereas GAD67 is detected exclusively in the high speed
supernatant. This data suggest that a pool GAD65, but not GAD67, is
associated with membrane compartments. After Triton X-114 extraction
and phase separation, a pool of both soluble and particulate GAD65
partitions in the detergent phase, whereas soluble GAD67 is recovered
only in the aqueous phase(14, 15) . The hydrophobicity
of GAD65 results from two hydrophobic post-translational modifications,
both of which occur at its NH-terminal region, (14) a domain where GAD65 differs significantly from GAD67. One
modification consists of the thiopalmitoylation of cysteines at
positions 30 and 45(16) , whereas the other has not yet been
characterized.
Immunocytochemical studies using antibodies which
recognize both GAD isoforms or exclusively GAD65 demonstrated that GAD
is localized in close proximity of neuronal synaptic vesicles (SVs) (17, 18) and -cell synaptic-like microvesicles
(SLMVs)(18) , the organelles involved in the storage and
secretion of non-peptide classical neurotransmitters such as
GABA(19, 20) . A pool of GAD65 is also concentrated in
the Golgi complex region of neurons, pancreatic
-cells, and
transfected CHO and COS cells(15, 21) . Targeting of
GAD65 to the Golgi complex region(19) , requires a signal
located at its NH
terminal region, but not its
palmitoylation(16, 21) . In contrast to GAD65, GAD67
was reported to be a soluble cytosolic protein both in the pancreas (14) and in transfected fibroblasts(15) . A fraction of
GAD67 was found to be concentrated in nerve terminals (3) ,
reflecting an interaction with other cell components, possibly GAD65
itself, since GAD65 and GAD67 were reported to form dimers and
heterodimers(11) .
In this study we investigated the
possible interaction of GAD67 with GAD65 in transfected fibroblasts as
well as in neurons and pancreatic -cells. Our data demonstrate
that the two proteins are associated and that because of its
interaction with GAD65 also GAD67 is targeted to intracellular membrane
compartments.
Figure 1:
A, distribution of GAD67 and GAD65 in
crude rat brain subcellular fractions and Triton X-114
temperature-induced phases. PNS, HSP, HSS, sol, det, and aq correspond to postnuclear
supernatant, high speed pellet, high speed supernatant, Triton X-114
soluble extract of postnuclear supernatant, Triton X-114 detergent
phase, and Triton X-114 aqueous phase, respectively. A pool of GAD67 is
recovered in the high speed pellet (lane 2) and in the Triton
X-114 detergent phase (lane 5). B, distribution of
GAD67 and GAD65 following differential subcellular fractionation and
Triton X-114 extraction of rat brain homogenates. P1, S1, P2, S2, P3, S3, and ins correspond to 1,000 g pellet, 1,000
g supernatant, 36,000
g pellet,
36,000
g supernatant, 170,000
g pellet, 170,000
g supernatant, and Triton X-114
insoluble material, respectively. A pool of GAD67 is recovered in each
pellet fraction (lanes 1, 11, and 21) and in Triton
X-114 detergent phases obtained from each pellet (4, 14, 24) and
supernatant(9, 19, 29) . Western blots in A and B were performed using serum
7673.
Figure 2: Immunoprecipitation of GAD67 and GAD65 from transfected COS cells and rat brain with anti-GAD65 specific antibody GAD6. GAD65 COS, GAD67 COS, GAD67 + GAD65 COS correspond to Triton X-100 extracts from COS cells transfected with GAD65, GAD67, or both GAD67 and GAD65, respectively. GAD67 COS + GAD65 COS corresponds to a mixture (1:1) of the Triton X-100 extracts of COS cells independently transfected with GAD67 or GAD65; rat brain, rat brain postnuclear supernatant. Levels of GAD65 and GAD67 expression in single (lanes 1 and 2, respectively) and co-transfected (lane 3) COS cells were comparable, as shown by Western blot with serum 7673 (lanes 1-3). Lanes 4-7 show GAD6 immunoprecipitates as visualized by Western blot with serum 7673. GAD6 co-immunoprecipitated both isoforms from brain (lane 4) and co-transfected COS cells (lane 7), but not from GAD67 COS (lane 5) or the mixture of extracts from GAD67 COS and GAD65 COS (lane 6).
Figure 3: Characterization of serum 9886 by Western blot and immunofluorescence. Top panel, Western blot on rat brain postnuclear supernatant with serum 7673 (lane 1), serum 9886 (lane 2), and GAD6 (lane 3). Bottom panel, immunofluorescence with serum 9886 on GAD67 transfected CHO cells (A) and GAD65 transfected CHO cells (B). Serum 9886 recognizes GAD67, but not GAD65. GAD67 in single transfected CHO cells appears evenly distributed in the cytosol. Bar, 20 µm.
Figure 4: Localization of GAD67 in GAD67 + GAD65-co-transfected CHO cells. Double immunofluorescence with serum 9886 (A, C), GAD6 (B), and fluorescein-conjugated lentil lectin (D). In GAD67 + GAD65 co-transfected CHO cells, a pool of GAD67 was co-localized with GAD65 in the region of the Golgi complex. Bar, 24 µm.
Figure 5:
Localization of GAD67 in GAD67 +
GAD65(1-83)/galactosidase-, GAD67 +
-galactosidase-, and GAD67 + GAD65
(S1-6)-co-transfected CHO cells. Double immunofluorescence with
serum 9886 (A, C, E), anti-
-galactosidase (B,
D), and fluorescein-conjugated lentil lectin (F). A pool
of GAD67 was localized in the region of the Golgi complex when
co-transfected in CHO cells with GAD65(1-83)/
-galactosidase
and GAD65 (S1-6). In GAD67 +
-galactosidase
co-transfected CHO cells, GAD67 was evenly distributed in the cytosol. Bar, 21 µm.
Figure 6: Localization of GAD67 and GAD65 in rat GABA-ergic neurons of the nucleus reticularis thalami. Double channel confocal microscopy for GAD67 (pseudocolor red) and GAD65 (pseudocolor green) with serum 9886 and GAD6, respectively. A pool of GAD67 was co-localized (pseudocolor yellow) with GAD65, in a perinuclear region corresponding to the Golgi complex (arrow) and in many nerve terminals (arrowheads) of GABA-ergic neurons. Bar, 19 µm.
Immunofluorescence in rat pancreatic islets
demonstrated that the expression of GAD67 and GAD65 (6) was restricted to
-cells. GAD67 was detected in all
-cells (Fig. 7C),
although in the
majority of the cells immunoreactivity for GAD65 was predominant (Fig. 7A). On the other hand, a few
-cells
strongly positive for GAD67 (Fig. 7, A and B,
arrowheads) were negative for GAD65 (Fig. 7C,
arrowheads). In some
-cells the two proteins appeared
co-localized in the perinuclear region (Fig. 7A,
arrow), presumable in correspondence of the Golgi apparatus.
Figure 7:
Localization of GAD67 and GAD65 in rat
pancreatic islets. Double (A) and single channel (B,
C) confocal microscopy for GAD67 (A and B, pseudocolor red) and GAD65 (A and C, pseudocolor green) with serum 9886 and GAD6, respectively.
GAD65 immunoreactivity was higher than that of GAD67 in most
-cells. In cells positive for GAD65 and GAD67, the two proteins
were co-localized in the perinuclear region (A, arrow). In a
few
-cells strongly positive for GAD67 (A and B,
arrowheads), GAD65 was not detectable (C, arrowheads). Bars, 13 µm (A) and 16 µm (B and C).
Previous studies have indicated GAD may exist as a dimer
(reviewed in (36) ). GAD was detected as a doublet with
molecular weight of 120,000 and 115,000 ± 5,000 (37) following SDS-gel electrophoresis of rat brain homogenates
in nonreducing conditions, suggesting that GAD isoforms may associate
in a homodimeric complex. Other evidence suggested that GAD forms
heterodimers(11, 23) , but these experiments did not
examine whether the two proteins interact in vivo. Here, we
definitively demonstrate that GAD67 binds to GAD65 in vivo.
Our results indicate that GAD67 interacts with both the soluble and the
particulate forms of GAD65 and that this interaction has implications
for the targeting of GAD67. We have also shown that GAD67, in agreement
with our previous suggestion(11) , binds to the
NH-terminal region of GAD65, a region with low homology to
the corresponding region of GAD67 and which contains the information
required for the targeting to intracellular membrane compartments (14, 15, 16, 21) . In addition, we
have demonstrated that preventing the palmitoylation of GAD65 does not
abolish its interaction with GAD67. In particular, GAD67 binds to GAD65
(S1-6), a GAD65 construct in which all 6 cysteines at the
NH
-terminal region were mutagenized (21) ,
indicating that formation of GAD65-GAD67 complexes is not due to
disulfide bridges, as reported previously(38) . Although the
presence of disulfide bridges between the two GAD isoforms in
nonreducing conditions is clearly an artifact generated by exposing the
two proteins to an oxidative environment upon homogenization, this
artifact clearly reflects the close association of the two proteins in vivo.
As a result of its association with GAD65, a pool
GAD67 is targeted to the Golgi complex region in double-transfected CHO
cells as well as in GABA-ergic neurons and in rat pancreatic
-cells. Analysis of rat pancreatic
-cells by confocal
microscopy has shown previously that a pool of GAD is concentrated in
the Golgi complex region(39) . Those experiments, however, were
performed using a serum (serum 1440) (40) which recognizes both
GAD isoforms and therefore could not address the molecular mechanism/s
responsible for the targeting of either isoforms to membrane
compartments. Recovery of GAD67 in the P3 pellet following differential
subcellular fractionation of rat brain homogenates strongly suggests
that a pool of the protein is associated with SVs, and by analogy, with
SLMVs of pancreatic
-cells. This association is most likely
mediated by its interaction with GAD65. Consistent with this idea,
synaptic terminals were found to contain either both proteins or GAD65,
but never GAD67 in the absence of GAD65 ( (35) and this study).
The interaction of GAD with SVs and SLMVs may be instrumental for the
rapid uptake of the newly synthesized GABA into these secretory
organelles(18, 19) .
Biochemical analysis suggests
that GAD67 is differentially distributed in rat brain and pancreatic
islets. Upon subcellular fractionation and Triton X-114 extraction of
brain homogenates, a pool of GAD67 is found in pellets and in Triton
X-114 detergent phases (23 and this study). In contrast, GAD67 from
pancreatic islets was reported to be present in the high speed
supernatant and in the Triton X-114 aqueous
phase(13, 14) . These results can be reconciled,
however, when the different levels of GAD67 and GAD65 co-expressed in
GABA-ergic neurons and pancreatic -cells are taken into account.
The high level of both GAD isoforms in most GABA-ergic neurons ( (35) and this study) allows GAD65-GAD67 complexes to be easily
detected, which, in turn, explains the partial recovery of GAD67 in
pellet and hydrophobic fractions. On the other side, the low amount of
GAD67 in rat pancreatic islets, together with its limited co-expression
with GAD65 within the same
-cells, may explain the virtual absence
of GAD67 in pellet and hydrophobic fractions of rat pancreatic islets.
Previous studies have emphasized the different biochemical
properties of GAD65 and GAD67 with implication for their intracellular
targeting. In this study we have demonstrated that, in spite of their
differences, a substantial pool of the two proteins co-localizes in
vivo due to their association. It has been proposed that the
differential interaction of GAD67 and GAD65 with pyridoxal
5`-phosphate, the co-factor required for GAD activity, provides a
mechanism for modulating GABA synthesis at nerve
terminals(3, 4, 5) . The GABA content of
neuronal synaptic vesicles and -cell synaptic like microvesicles
may be further controlled by regulating the association of GAD65 with
membranes and the interaction of GAD67 with GAD65. In this context it
is interesting that the NH
-terminal region of GAD65 is
involved in membrane association as well as in binding to GAD67.
The physiological significance of the concentration of GAD67 and GAD65 in the region of the Golgi complex remains to be established. The most likely possibility is that the targeting of the two proteins to the Golgi compartment is an intermediate step in their route toward SVs and SLMVs. GAD oligomers may associate at this stage with precursors of SVs and SLMVs which bud from the Golgi complex and are then directed to the cell periphery(41) . Targeting to the Golgi complex region may also be required for post-translational modifications of GAD65(21) . Palmitoylation increases the hydrophobicity of GAD65 and might further strengthen the association of both GAD65 and GAD65-GAD67 complexes to membrane organelles, including SVs and SLMVs.