©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeting of the 67-kDa Isoform of Glutamic Acid Decarboxylase to Intracellular Organelles Is Mediated by Its Interaction with the NH-terminal Region of the 65-kDa Isoform of Glutamic Acid Decarboxylase (*)

(Received for publication, September 16, 1994; and in revised form, November 18, 1994)

Ronald Dirkx Jr. (1) Annette Thomas (2) Linsong Li (3) Åke Lernmark (3) (4) Robert S. Sherwin (1) Pietro De Camilli (2) Michele Solimena (1)(§)

From the  (1)Department of Internal Medicine, Section of Endocrinology, the (2)Department of Cell Biology, and the Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, the (3)Department of Medicine, University of Washington, Seattle, Washington 98195, and the (4)Department of Endocrinology, Karolinska Institute, Karolinska Hospital L1:02, S-171 76 Stockholm, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter -aminobutyric acid in neurons and pancreatic beta-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 beta-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 beta-cells. GAD67 interacts with the NH(2)-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.


INTRODUCTION

Glutamic acid decarboxylase (GAD) (^1)synthesizes the neurotransmitter -aminobutyric acid (GABA) in neurons and pancreatic beta-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(2)-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 beta-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 beta-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(2)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 beta-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.


MATERIALS AND METHODS

Antibodies and Cytochemical Probes

An anti-GAD67 specific antiserum (serum 9886) was raised by immunizing a rabbit with a peptide corresponding to amino acids 2-19 of GAD67(22) , a region of the protein which has no homology with GAD65. Rabbit serum 7673, which recognize both GAD isoforms, was raised against a peptide corresponding to amino acids 577-593 of rat GAD67(15) . The GAD65-specific monoclonal antibody GAD6 (23) was a gift from Dr. D. Gottlieb (Washington University, St. Louis, MO). A commercial monoclonal antibody against beta-galactosidase was purchased from Promega (Madison, WI). Fluorescein isothiocyanate-conjugated lens culinaris was purchased from E-Y Laboratories (San Mateo, CA). Secondary antibodies conjugated to fluorophores (fluorescein and lissamine rhodamine) were purchased from Boehringer Mannheim (Indianapolis, IN).

Transfection of GAD Constructs in COS and CHO Cells

The cloning of a rat GAD65 cDNA has been reported previously(15) . The rat GAD67 cDNA was a gift from Dr. D. Gottlieb(24) . The GAD67 and GAD65 constructs in the pRC/RSV expression vector (Invitrogen, San Diego, CA) have been described previously(15, 21) . COS-7 and CHO cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 34 µg/ml proline. GAD constructs in the pRC/RSV expression vector were transiently transfected into COS cells by a Ca-phosphate procedure(25, 26) . Transfection of CHO cells using Lipofectin reagent (Life Technologies, Inc.) was performed as described previously(15) . Co-transfections were carried out using a mixture containing 15 µg of each plasmid DNA.

Rat Brain Subcellular Fractionation and Triton X-114 Assay

All biochemical procedures were performed at 4 °C, unless otherwise specified. Rat brain homogenates prepared in homogenization buffer (HB) (150 mM NaCl, 10 mM HEPES buffer, pH 7.4, 100 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, and 1 µg/ml of leupeptin, antipain, pepstatin, and aprotinin) were spun at 3,000 times g for 10 min. 1-ml aliquots of postnuclear supernatant were further centrifuged at 100,000 times g for 20 min in a TLA 100.2 rotor using a Beckman tabletop ultracentrifuge (Beckman, Palo Alto, CA) to generate a high speed pellet and supernatant. For Triton X-114 extraction assays, aliquots of the postnuclear supernatant were extracted with 2% precondensed Triton X-114 (Pierce) for 90 min. Triton X-114-insoluble material was removed by centrifugation at 15,000 times g for 10 min and resuspended in HB with 2% Triton X-114 to the original volume. The soluble material was phase-partitioned at 37 °C for 5 min as described previously(15, 27) . Additionally, rat brain homogenates prepared as described above were first spun at 1,000 times g for 10 min to generate a P1 pellet and a S1 supernatant. Centrifugation of S1 at 36,000 times g for 30 min in a SS-34 rotor (Sorvall, Boston, MA) generated a P2 pellet and a S2 supernatant. Centrifugation of S2 at 170,000 times g for 2 h in a 50 Ti Beckman rotor produced a P3 pellet and a S3 supernatant. Each pellet was brought back to the original volume in HB. Aliquots of all fractions were solubilized with 2% Triton X-114 and phase-partitioned as described above. Equal volumes of each subcellular fraction and Triton X-114 extracts were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose according to standard procedures(28, 29) . Immunoblotting was performed using serum 7673, serum 9886, and GAD6 ascites at dilutions of 1:250. Bound antibodies were detected using peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (1:5000) (Sigma) followed by enhanced chemiluminescence (ECL) reagent (Amersham Corp.). Alternatively, rabbit immunoglubulins were revealed by I-protein A radioimmunolabeling (10^5 cpm/ml) (Amersham) and autoradiography.

Immunoprecipitations

Seventy-two hours after transfection, COS cells were solubilized with 1 ml of HB containing 2% Triton X-100 (Sigma) for 90 min. One confluent 10-cm dish of transfected cells was used per immunoprecipitation. Insoluble material was removed by centrifugation 15,000 times g for 10 min. In some experiments Triton X-100 extracts from GAD67- and GAD65-COS cells were mixed in a 1:1 ratio and used as a starting material for immunopreciptation. The Triton X-100-soluble material was precleared with 125 µl of 50% protein A-Sepharose CL-4 beads (Pharmacia Biotech Inc.) for 1 h. Samples were then centrifuged at 750 times g for 1 min, and the resulting supernatants were used as a starting material for immunoprecipitation with 10 µl of GAD6 ascites as described previously(18) . Immunoprecipitates were separated by SDS-PAGE and immunoblotted with serum 7673 as described above.

Immunofluorescence and Confocal Microscopy

Double immunofluorescence on transfected CHO cells and 10-µm cryostat sections of rat brain or pancreas was performed as described previously (30, 31) . Dilution of primary antibodies were as follows: serum 9886, 1:100; GAD6 ascites, 1:50; beta-galactosidase, 1:100. Fluorescein-conjugated lentil lectin was used at 20 µg/ml. Confocal microscopy was performed using a Bio-Rad MRC 600 system (Bio-Rad) equipped with an argon ion laser connected to a Zeiss Axiovert microscope (Zeiss, Thornwood, NY). Acquired images were processed as described previously(32) .


RESULTS

A Pool of Rat Brain GAD67 Is Recovered in Pellet Fractions and in Triton X-114 Detergent Phases

After centrifugation of rat brain postnuclear supernatant at 100,000 times g, both GAD isoforms were partially recovered in the high speed pellet (Fig. 1A, lane 2), indicating that in neurons not only GAD65, but also GAD67, may be associated with membranes, as suggested previously (23) . To rule out the possibility that sedimentable GAD67 corresponds only to a pool of the soluble enzyme entrapped in synaptosomes or other resealed cell fragments, rat brain homogenates were further analyzed by differential subcellular fractionation. As shown in Fig. 1B, GAD67 was recovered together with GAD65 in all pellet fractions (P1, P2, P3), including P3 (lane 21), which contains only small vesicles including SVs, and virtually no soluble proteins(33) . Moreover, following Triton X-114 extraction a significant amount of GAD67 from postnuclear supernatant partitioned in the detergent phase (Fig. 1A, lane 5). A similar partitioning of GAD67 was observed in Triton X-114 extracts of all pellet (Fig. 1B, lanes 9, 19, and 29) and supernatant (Fig. 1B, lanes 4, 14, and 24) fractions obtained by differential centrifugation of brain homogenates.


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 times g pellet, 1,000 times g supernatant, 36,000 times g pellet, 36,000 times g supernatant, 170,000 times g pellet, 170,000 times 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.



Interaction between GAD67 and GAD65 Requires Their Co-expression in Living Cells

The recovery of GAD67 together with GAD65 in pellet fractions and detergent phases may be due to an interaction of GAD67 with GAD65, because the two proteins were reported to co-immunoprecipitate(11, 23) . To investigate the interaction between GAD67 and GAD65, we performed immunoprecipitation experiments from Triton X-100 extracts of COS cells transiently transfected with GAD67, GAD65, or both isoforms using the anti-GAD65 specific antibody GAD6. Levels of expression of GAD65 and GAD67 in single (Fig. 2, lanes 1 and 2) and double-transfected (Fig. 2, lane 3) COS cells were comparable, as shown by Western blot using serum 7673, which recognizes both proteins equally. As reported previously(11, 23) , GAD6 was able to immunoprecipitate GAD67 together with GAD65 from rat brain extracts (Fig. 2, lane 4) but not from COS cells transfected with GAD67 only (Fig. 2, lane 5). However, GAD6 co-immunoprecipitated GAD67 together with GAD65 from extracts of COS cells co-transfected with the two isoforms (Fig. 2, lane 7). Conversely, GAD6 did not co-immunoprecipitated GAD67 with GAD65 when GAD67 COS and GAD65 COS cell extracts were mixed and used as a starting material for immunoprecipitation (Fig. 2, lane 6). Thus, the association of GAD67 with GAD65 occurs before extraction, i.e. within living cells expressing both proteins. Following affinity purification from rat brain extracts on a GAD6 column, no bands other than GAD65 and GAD67 were visualized in the eluate by Coomassie Blue staining after SDS-PAGE (23) . (^2)This finding suggests GAD67 and GAD65 interact directly and not via another protein.


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).



Formation of GAD65-GAD67 Complexes Results in the Targeting of GAD67 to the Golgi Apparatus

Next, we used a GAD67 specific antiserum (serum 9886) (22) to compare the intracellular distribution of GAD67 when expressed alone or together with GAD65 in CHO cells. Serum 9886, unlike other antibodies used until now for the immunocytochemical detection of GAD67(3) , recognizes exclusively GAD67, as shown by Western blot on rat brain postnuclear supernatants (Fig. 3A, lane 2) and by immunocytochemistry on CHO cells transfected either with GAD67 (Fig. 3B, left panel) or GAD65 (Fig. 3B, right panel). In CHO cells transfected only with GAD67, this protein appeared homogeneously distributed in the cytosol, as expected for a soluble protein and as reported previously(15) . In GAD67 GAD65 co-transfected CHO cells, however, a pool of GAD67 (Fig. 4, A and C) was concentrated together with GAD65 (Fig. 4B) in a region of the Golgi apparatus, as shown by its co-localization with lentil lectin (Fig. 4D). Lentil lectin binding sites are concentrated in the Golgi complex(34) . Therefore, upon co-expression and interaction with GAD65, GAD67 is targeted to intracellular vesicular compartments.


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.



GAD67 Binds to the NH(2)-terminal Region of GAD65

We next mapped the domain of GAD65 responsible for the interaction with GAD67. In previous experiments we found that only two anti-GAD sera, N65 and 9057, among a large number tested, were not able to co-immunoprecipitate GAD67 with GAD65 from rat brain extracts. Both N65 and 9057 sera contain antibodies directed against epitopes localized within amino acids 1-95 of GAD65. The lack of GAD67 in immunoprecipitates produced by these antibodies suggested that their epitope(s) is(are) hidden in the GAD65-GAD67 complex, i.e. that the NH(2)-terminal region of GAD65 may be involved in binding GAD67. To test this hypothesis GAD67 was co-transfected in CHO cells either with beta-galactosidase or with GAD65(1-83)/beta-gal, a fusion protein in which amino acids 1-83 of GAD65 are fused to the NH(2)-terminus of beta-galactosidase. We have shown previously that in transfected CHO cells, beta-galactosidase is evenly distributed in the cytosol, whereas GAD65(1-83)/beta-gal is concentrated in the Golgi complex region(21) , thus demonstrating that amino acids 1-83 of GAD65 contain a signal which is sufficient to target an unrelated soluble protein to this intracellular compartment. Upon co-transfection with GAD65(1-83)/beta-gal, GAD67 (Fig. 5A) co-localized with the GAD65/beta-gal chimera (Fig. 5B) in the region of the Golgi complex, indicating that amino acids 1-83 of GAD65 contain the GAD67-binding domain. On the other hand, GAD67 was diffusely distributed (Fig. 5C) when co-transfected with beta-galactosidase (Fig. 5D). In addition, a pool of GAD67 (Fig. 5E) was concentrated in the Golgi complex region when co-transfected in CHO cells with GAD65(S1-6), a GAD65 mutant which cannot undergo thiopalmitoylation (21) because of the site-directed mutagenesis of all cysteines within its NH(2)-terminal region. Therefore, NH(2)-terminal palmitoylation of GAD65 is not required for association of the two proteins.


Figure 5: Localization of GAD67 in GAD67 + GAD65(1-83)/betagalactosidase-, GAD67 + beta-galactosidase-, and GAD67 + GAD65 (S1-6)-co-transfected CHO cells. Double immunofluorescence with serum 9886 (A, C, E), anti-beta-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)/beta-galactosidase and GAD65 (S1-6). In GAD67 + beta-galactosidase co-transfected CHO cells, GAD67 was evenly distributed in the cytosol. Bar, 21 µm.



Partial Co-localization of GAD67 and GAD65 in Rat GABA-ergic Neurons and Pancreatic beta-Cells

Double immunostaining of rat brain sections with serum 9886 and GAD6 demonstrated that both GAD67 and GAD65 are abundant in nerve terminals. As described previously (3, 35) GAD67 appeared slightly enriched in the perikaryal-dendritic region of GABA-ergic neurons (Fig. 6), (^3)whereas GAD65 is more enriched than GAD67 in synapses. In addition, we observed that in neuronal cell bodies, as in GAD65-GAD67 co-transfected CHO cells, a pool of GAD67 was co-localized with GAD65 in the Golgi complex region (Fig. 6, arrow). Nerve terminals were either positive for both isoforms (Fig. 6, arrowheads) or exclusively contained GAD65, whereas no synaptic terminals appeared positive for GAD67 in the absence of GAD65.


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 GAD67^3 and GAD65 (6) was restricted to beta-cells. GAD67 was detected in all beta-cells (Fig. 7C),^3 although in the majority of the cells immunoreactivity for GAD65 was predominant (Fig. 7A). On the other hand, a few beta-cells strongly positive for GAD67 (Fig. 7, A and B, arrowheads) were negative for GAD65 (Fig. 7C, arrowheads). In some beta-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 beta-cells. In cells positive for GAD65 and GAD67, the two proteins were co-localized in the perinuclear region (A, arrow). In a few beta-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).




DISCUSSION

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(2)-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(2)-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 beta-cells. Analysis of rat pancreatic beta-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 beta-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 beta-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 beta-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 beta-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(2)-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.


FOOTNOTES

*
This work was supported by a Juvenile Diabetes Foundation International Postdoctoral Fellowship (to M. S.) and a Juvenile Diabetes Foundation International Career Development Award (to M. S.), by National Institutes of Health Grants DK-45735 (to M. S.) and DK-43078 (to P. D. C.), and by a Research Project Award from the McKnight Endowment for the Neurosciences (to P. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8020. Tel.: 203-737-1129; Fax: 203-785-6015.

(^1)
The abbreviations used are: GAD, glutamic acid decarboxylase; GABA, -aminobutyric acid; CHO cells, Chinese hamster ovary cells; SVs, synaptic vesicles; SLMVs, synaptic-like microvesicles; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Butler, R. Dirkx, Jr., P. De Camilli, and M. Solimena, unpublished observations.

(^3)
R. Dirkx, Jr., P. DeCamilli, and M. Solimena, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. M. Butler for materials and advice, Dr. S. Artavanis-Tsakonas and L. Caron for access to and assistance with confocal microscopy, L. Daniel for assistance with cryostat sectioning and immunofluorescence, Dr. P. Hanson for assistance and advice in double transfection experiments, and K. Conlin for technical support. We also thank Dr. D. Gottlieb for the generous gift of the monoclonal antibody GAD6 and of the rat GAD67 cDNA.


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