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J. Biol. Chem., Vol. 275, Issue 27, 20822-20828, July 7, 2000

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Association of L-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles^*

Che-Chang Hsu, Kathleen M. Davis, Hong Jin, Todd Foos, Erik Floor, Weiqing Chen, John B. Tyburski, Chao-Yuh Yang^§, John V. Schloss^¶, and Jang-Yen Wu^**

From the Departments of  Molecular Biosciences and ^¶ Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, the ^§ Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, and the  Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Received for publication, February 21, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently we have reported that the membrane-associated form of the -aminobutyric acid-synthesizing enzyme, L-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366-24371). In this report, several lines of evidence are presented to indicate that L-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD₆₅ as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD₆₅-specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD₆₅ and GAD₆₇ are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in -aminobutyric acid neurotransmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

L-Glutamate decarboxylase (GAD¹; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of -aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian brain (1). There are two well characterized GAD isoforms in the brain, namely GAD₆₅ and GAD₆₇, referring to GAD with molecular masses of 65 and 67 kDa, respectively (for review, see Ref. 2). GAD₆₇ is mostly soluble and is distributed evenly throughout the cell, whereas GAD₆₅ is concentrated at the nerve terminals (3) and constitutes the majority of the membrane-associated GAD (MGAD) (4, 5). Despite its importance, our knowledge regarding the regulation of GAD activity is quite limited. Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by a Ca²⁺-dependent phosphatase, calcineurin, and is inhibited by phosphorylation, mediated by a cAMP-dependent protein kinase A (6, 7). Conversely, MGAD is activated by protein phosphorylation, which depends on the integrity of the electrochemical gradient of synaptic vesicles (5). Hence, GAD activity appears to be regulated differently depending on whether it exists as a soluble or membrane-anchored protein. Judging from the amino acid sequences, it is unlikely that GAD₆₅ and/or GAD₆₇ can be integral membrane components, since neither contains a stretch of hydrophobic amino acids long enough to span the membrane (greater than 20 residues), a typical feature for integral membrane proteins. Furthermore, both isoforms lack the appropriate consensus sequences for the attachment of GAD to membranes through fatty acylation via esterification, or N-myristoylation (2).

Now evidence is presented here to show that GAD can become membrane-associated through interactions with a member of the heat shock protein 70 family, heat shock cognate 70 (HSC70), which is then anchored to synaptic vesicles through interactions with an intrinsic synaptic vesicle protein, cysteine string protein (CSP). In addition, a model is also proposed to show the anchoring mechanism of GAD to synaptic vesicles and a functional link between GABA synthesis and vesicular GABA transport at the nerve terminals.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Fresh porcine brains were obtained from a local abattoir. Bovine brain HSC70 and biotinylated HSC70 were obtained from StressGen Biotechnologies Corp. (Collegeville, PA). Anti-GAD₆₅ and anti-GAD₆₇ are polyclonal rabbit antibodies raised against recombinant human GAD₆₇ (HGAD₆₇) and human GAD₆₅ (HGAD₆₅) expressed in separate bacterial systems (8). Subtype-specific polyclonal antibodies of GAD₆₅ and GAD₆₇ were prepared by preadsorbing anti-HGAD₆₅ serum with an excess of recombinant HGAD₆₇ to remove GAD₆₇-specific antibodies. Similarly, the specific anti-HGAD₆₇ serum was obtained by pretreatment of anti-HGAD₆₇ serum with an excess of recombinant HGAD₆₅ as described previously (8). GAD6, a GAD₆₅-specific monoclonal antibody, was purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Rabbit polyclonal anti-HSP70 sera were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-CSP rabbit sera were purchased from Chemicon International, Inc. (Temecula, CA). Benzethonium hydroxide (hyamine base, 1 M solution in methanol), pyridoxal 5'-phosphate (PLP), 2-aminoethylisothiuronium bromide (AET), dithiothreitol, ATP, AMP-PNP, Nycodenz, and Triton X-100 were purchased from Sigma. Ceramic hydroxylapatite, acrylamide, bisacrylamide, tetramethylenediamine, ammonium persulfate, -mercaptoethanol, and protein assay kit were purchased from Bio-Rad. [L-¹⁴C]Glutamic acid was purchased from NEN Life Science Products, and Western-Lite Plus immunodetection kit was purchased from Tropix Inc. (Bedford, MA). HiTrap protein A columns, N-hydroxysuccinimide-activated Sepharose columns, and ECL reagent were purchased from Amersham Pharmacia Biotech. All other chemicals were of the purest grade commercially available.

Enzyme Assay-- GAD was assayed by a radiometric method measuring the formation of ¹⁴CO₂ from [L-¹⁴C]glutamic acid as described previously (9).

Preparation of Synaptosomal Membranes-- Unless mentioned otherwise, all purification procedures were carried out at 4 °C, and in standard GAD buffer containing 50 mM potassium phosphate, 1 mM AET, 0.2 mM PLP at pH 7.2. In a typical preparation, a 15% (w/v) porcine brain homogenate was made with a glass-Teflon homogenizer in standard GAD buffer containing 1 mM MgCl₂, 1 mM phenylmethysulfonyl fluoride, 5 mM benzamidine, and 1 mM theophylline. Preparation of crude synaptosomes was performed as described previously (6, 7). Briefly, fresh porcine brains were homogenized in 0.32 M sucrose (w/v, 15 g/100 ml), and the homogenate was centrifuged at 1,000 × g for 10 min to remove cell debris and the nuclear fraction. The post-nuclear supernatant solution was centrifuged at 23,000 × g for 30 min, and the pellet thus obtained was the crude synaptosomal fraction referred to as P₂.

Purification of MGAD-- Purification of MGAD was conducted, as described previously with slight modifications (10-12). The P₂ fraction obtained from the preceding step was first lysed in GAD buffer, followed by centrifugation at 100,000 × g for 1 h. The liquid thus obtained was referred to as supernatant (S1). The resulting pellet, P₂M, was solubilized with 0.5% Triton X-100 in standard GAD buffer solution. The solubilized MGAD was further purified through conventional column chromatography consisting of an anion exchange (DEAE-52), an adsorption (ceramic hydroxylapatite), and a gel filtration column (Sephadex G-200). In addition to the conventional column procedures, MGAD was also purified by anti-GAD immunoaffinity column as described previously (13) with slight modifications. Specific anti-GAD IgG was prepared from anti-GAD sera using protein A column chromatography. Briefly, anti-GAD sera were first loaded on protein A columns, followed by washing with eight column volumes of buffer containing 20 mM sodium phosphate at pH 7. The column was then eluted with six column volumes of buffer containing 0.1 M sodium citrate, pH 3.0. Fractions were neutralized to pH 7.0 with pre-titrated amount of 1 N NaOH. Anti-GAD IgG thus purified was coupled to N-hydroxysuccinimide-activated Sepharose according to the manufacturer's instructions (Amersham Pharmacia Biotech). Anti-GAD IgG immunoaffinity columns were then used for purification of GAD and GAD-associated protein complex as detailed in the following section.

Preparation of Recombinant Human Brain GAD₆₅ and GAD₆₇-- Recombinant human brain GAD₆₅ and GAD₆₇, referred to as HGAD₆₅ and HGAD₆₇, respectively, were prepared as described recently (8). Briefly, HGADs were overexpressed in DH5 Escherichia coli, which were transformed with either a HGAD₆₅ or HGAD₆₇ recombinant pGEX-3X plasmid, as glutathione S-transferase (GST) fusion proteins. GST-HGAD fusion proteins were first purified by glutathione affinity column chromatography, followed by cleavage of the fusion proteins with factor Xa. Final purification of free HGAD₆₅ and HGAD₆₇ were achieved using repetitive glutathione affinity columns (8).

In Vitro Reconstitution Assay Using Non-denaturing Gradient Polyacrylamide Gel Electrophoresis (NG-PAGE)-- Biotinylated bovine HSC70 (2 µg) was incubated at 37 °C for 1 h with either HGAD₆₅ (2 µg) or HGAD₆₇ (2 µg) in a final volume of 10 µl of 50 mM Tris-citrate (pH 7.2). The resulting protein complexes were analyzed by electrophoresis on 5-25% NG-PAGE under nonreducing conditions. Running conditions were modified from the procedure described previously (14). One volume of the protein sample was mixed with two volume of sample buffer containing 50 mM Tris-HCl, pH 6.8, 500 mg/ml glycerol, and 2.5 mg/ml bromphenol blue. The samples were loaded on to a stacking gel, containing 0.5 M Tris-HCl, pH 6.8, 5% acrylamide/bisacrylamide. The separating gel contained a 5-25% gradient of acrylamide/bisacrylamide with 1.5 M Tris-HCl, pH 8.8. Electrophoresis was carried out for 8 h at 150 V of constant voltage in a running buffer consisting of 50 mM Tris and 200 mM glycine at pH 8.8, followed by transfer of proteins to polyvinylidene difluoride membranes. Complexes of biotinylated HSP70 and GAD proteins on polyvinylidene difluoride membranes were visualized either with alkaline phosphatase-conjugated streptavidin using Western-Lite Plus (Tropix Inc.) or by isoform-specific anti-GAD antibody and the ECL kit (Amersham Pharmacia Biotech).

Purification of Synaptic Vesicles-- Synaptic vesicles were purified from rat brains as described previously (15). Briefly, synaptic vesicles isolated from the microsomal fraction by density gradient (10-30% Nycodenz gradient) centrifugation were further purified sequentially on Sephacryl S-1000 and CPG-3000 size-exclusion columns in buffer consisting of 0.16 M KCl, 5 mM NaHPO₄, pH 6.6, 1 mM EGTA, 0.02% NaN₃, 1 mM dithiothreitol, and saturated (~1 µM) 3,5-di-t-butyl-4-hydroxybenzyl ether. Final purification was achieved by equilibrium centrifugation at 100,000 × g on a 4-26% gradient of Ficoll (M_r ~ 400,000).

Co-immunoprecipitation of HSC70 with GAD₆₅-- Five hundred microliters of Triton X-100-solubilized and partially purified MGAD preparations from porcine brain P₂M sample were used as the starting material for immunoprecipitation. GAD samples (500 µl) were first precleared by incubation with 100 µl of 50% (v/v) protein A-Sepharose at 4 °C for 2 h. The supernatant solutions obtained after centrifugation at 1000 × g for 3 min were then incubated with 50 µl of anti-GAD₆₅ monoclonal antibody, GAD6, at 4 °C for 12 h. Protein A-Sepharose (50 µl) was added to each mixture and incubated at 4 °C for an additional 2 h. The mixture was then centrifuged at 1000 × g for 3 min. The resulting pellet was then washed six times in standard GAD buffer. Both the supernatant and pellet were assayed for GAD activity to ensure presence of GAD in the pellet. Immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with anti-HSP70. Quantitative densitometry analyses of immunoblots were made with the Quantity One^TM imaging densitometer (model GS-700; Bio-Rad).

Immunopurification of GAD Protein Complex-- Anti-GAD₆₅ and anti-GAD₆₇ immunoaffinity columns as described in the preceding section were used to purify GAD-associated protein complex, using crude brain extracts of S1 and solubilized P₂M fraction. In a typical experiment, 80 ml of S1 or P₂M extract (2 mg/ml) in Tris-citrate buffer (50 mM Tris-citrate, pH 7.0, 1 mM phenylmethysulfonyl fluoride, 5 mM sodium floride, and 5 mM benzamidine, 1 mM AET, 0.2 mM PLP) was recirculated into 1 ml of anti-GAD₆₅ or anti-GAD₆₇ immunoaffinity column at 4 °C for 8 h. The columns were washed with 80 column volumes of the same Tris-citrate buffer, except that the concentration of Tris-citrate was increased to 500 mM. The columns were further eluted with eight column volumes (8 ml) of elution buffer containing 0.1 M citric acid, pH 3.0, at 4 °C. A titrated amount of 1 N NaOH solution was placed in the collecting tubes to neutralize and bring the pH up to 7.0 immediately after each collection. Protein concentration in each fraction was monitored at 280 nm. The fractions containing the highest protein concentration were then pooled (~ 3 ml) and dialyzed twice with 1 liter of 5 mM Tris-citrate buffer, pH 7.0, containing 1 mM AET and 0.2 mM PLP at 4 °C. The dialyzed samples were concentrated with a SpeedVac concentrator (Savant Instruments, Inc.) to 500 µl and then analyzed by SDS-PAGE and immunoblots with anti-HSP70 as well as anti-CSP.

Effect of Protein Complex Formation on GAD Activity in in Vitro Reconstitution Studies-- Mixtures of 2 µl of highly purified HGAD₆₅ (1 µg/µl), 2 µl of HSC70 (1 µg/µl), and/or 5 µl of highly purified synaptic vesicles were incubated at 37 °C for 10 min with constant agitation. At the end of the incubation, the entire mixture was transferred to a test tube and GAD activity was determined.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- Unless mentioned otherwise, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide/bisacrylamide gels) was carried out under reducing conditions with the presence of 0.05% -mercaptoethanol in the sample buffer. The running conditions of SDS-PAGE were followed as described by Laemmli (16). Immunoblotting test was conducted as described (6, 7). Briefly, blotting was carried out at 4 °C for 18 h in a LKB 2005 transfer unit containing 25 mM Tris-HCl (pH 6.8), 0.192 M glycine, 0.5% SDS, and 20% methanol followed by overnight incubation with primary antibody at 4 °C and 2 h of secondary antibody incubation in room temperature. Immunocomplex was visualized using Western-Light Plus or ECL reagents. Quantitative densitometry analyses of the Coomassie Blue-stained protein bands of SDS-polyacrylamide gels were made with the Quantity One^TM imaging densitometer (model GS-700; Bio-Rad).

Amino Acid Sequencing-- Highly purified MGAD preparation was digested with trypsin, followed by separation of tryptic peptides on a C₁₈ reverse column using high pressure liquid chromatography as described previously (17). Selected peptides were then sequenced for 10-15 Edman degradation cycles on a Precise Sequencer (Applied Biosystems, Foster City, CA), according to the manufacturer's specifications. Protein sequence alignment was performed using the BLAST program, with searches of the SwissProt data bank.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Co-purification of HSC70 with Membrane-associated GAD-- The protein pattern of partially purified MGAD fractions after hydroxylapatite column chromatography is shown in Fig. 1A. During purification of MGAD, two proteins at the approximate molecular mass of 67 kDa were found to correlate with GAD activity (Fig. 1B) throughout the purification steps as shown in the highlighted box of Fig. 1A and lane 1 of Fig. 2. Partial amino acid sequencing identified HSC70 as a major constituent of these protein bands with the following sequence: HWPFVVVNDAGRPK, which matched in 13 out of 14 amino acid residues to the ⁸⁹HWPFMVVNDAGRPK¹⁰² stretch of HSC70 amino acid sequence that is analogously present across species of murine, human, and bovine. In addition, immunoblotting tests with anti-HSP70 (Fig. 2, lane 2), GAD6 (Fig. 2, lane 3), and anti-GAD₆₇ indicate that the higher band contained both HSP70 and GAD₆₇, whereas the lower band contains GAD₆₅. These immunoblots also illustrate that no cross-reactivity was detected between antibodies of HSC70 and GAD₆₅. Thus, these results indicate that HSC70, GAD₆₅, and GAD₆₇ are membrane-associated and may constitute a protein complex in vitro.

View larger version (69K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis of solubilized MGAD fractions from ceramic hydroxylapatite column. A, fractions of MGAD purified through the ceramic hydroxylapatite column were analyzed on SDS-PAGE. B, the corresponding GAD activity associated with each fraction is shown directly below. Co-purification of HSC70 and GAD is illustrated by a highlighted box containing two prominent bands of HSC70 and GAD65.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Protein analysis of highly purified MGAD preparation by SDS-PAGE and immunoblots using specific antibodies to HSP70 and GAD65. The fraction containing the highest GAD activity was obtained after three columns, namely DEAE-52, ceramic hydroxylapatite, and Sephadex G-200. Lane 1, protein pattern showing the presence of dual bands at around the approximate molecular mass of 67 kDa, as indicated by Coomassie Blue staining on SDS-polyacrylamide gel; lane 2, immunoblotting with anti-HSP70 showing the presence of HSP70; lane 3, immunoblotting with monoclonal anti-GAD65 indicating the presence of GAD65; lane 4, immunoblotting with polyclonal anti-GAD67 indicating the presence of both GAD65 and GAD67. These results reveal co-purification of HSC70 and isoforms of GAD65 and GAD67, as identified by a polyclonal anti-HSP70 (lane 2), a monoclonal anti-GAD65 antibody (lane 3), and a polyclonal anti-GAD67 (lane 4).

Protein Complex Formation of GAD₆₅ and HSC70 but Not of GAD₆₇ and HSC70-- To elucidate the possible protein interaction between HSC70, GAD₆₅, and GAD₆₇, we utilized reconstitution assays using purified HGAD₆₅, HGAD₆₇, and purified HSC70 (~90% pure). The HSC70·HGAD complexes were separated from free HSC70 and HGAD by NG-PAGE and analyzed by immunoblot. Results in Fig. 3 (lanes 2 and 3) show the formation of protein complex between HGAD₆₅ and HSC70, as indicated by the parallel gel mobility shift of HSP70·HGAD complex, recognized by GAD6 and the biotin-labeled HSC70. Without HSC70, GAD₆₅ migrated to a lower molecular weight position in the gel (Fig. 3, lane 1). HGAD₆₇ and HSC70 do not form a protein complex and thus do not share this parallel shift in gel mobility (Fig. 3, lanes 5 and 6). Unlike GAD₆₅, the mobility of GAD₆₇ in the gel is unaffected by the addition of HSC70 (Fig. 3, lanes 4 and 5). In addition, lack of immunostaining by anti-GAD₆₇ at the position of HSC70 (Fig. 3, lane 5) indicates that no cross-reactivity was detected between anti-GAD₆₇ and HSC70. These results suggest the possibility of protein complex formation specifically between HSC70 and HGAD₆₅, but not between HSC70 and HGAD₆₇.

View larger version (97K): [in this window] [in a new window]   Fig. 3.   Reconstitution assay of immunocomplex between HSC70 recombinant human GAD65 and GAD67 under non-denaturing gradient PAGE. Purified human recombinant GAD (HGAD65 or HGAD67, 2 µg each), were incubated with purified biotinylated bovine heat shock cognate 70 (HSC70, 2 µg) and analyzed on non-denaturing gradient PAGE. Lane 1, GAD65 probed with polyclonal anti-GAD65; lane 2, biotinylated HSC70 and GAD65 mixture probed with anti-GAD65 showing complex formation between HSC70 and GAD65 with little amount of free GAD65 remained; lane 3, biotinylated HSC70 and GAD65 mixture, showing complex formation between HSC70 and GAD65, identical to that of lane 2; lane 4, GAD67 probed with anti-GAD67; lane 5, biotinylated HSC70 and GAD67 mixture probed with anti-GAD67 showing just free GAD67 and no complex formation; lane 6, biotinylated HSC70 and GAD67 mixture shows the position of HSC70 protein aggregation alone without GAD67. Protein complex formation was observed with incubation of GAD65 and HSC70 but not with GAD67 and HSC70. GAD isoforms were visualized by polyclonal anti-GAD65 and anti-GAD67, as detected by ECL kit (Amersham Pharmacia Biotech). Biotinylated HSC70 was visualized by incubation with alkaline phosphatase-conjugated streptavidin and detected with Western-Lite Plus (Tropix, Inc.).

GAD₆₅·HSC70 Protein Complex Formation as Detected by Immunoprecipitation-- If GAD₆₅ does indeed form a protein complex with HSC70, one would expect the antibodies recognizing GAD₆₅ to also immunoprecipitate HSC70. Indeed, the GAD₆₅-specific monoclonal antibody, GAD6 (18), was able to immunoprecipitate GAD₆₅ in a highly purified porcine brain MGAD preparation. Under stringent washing conditions, the presence of HSC70 in the GAD·anti-GAD₆₅ immunoprecipitate was detected, as visualized by immunoreactivity to polyclonal anti-HSP70 (Fig. 4, lane C).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Immunoprecipitation of HSP70 with monoclonal anti-GAD65. Partially purified MGAD preparation, which contained HSC70 as shown in immunoblotting tests with anti-HSP70 (lane A), was immunoprecipitated with GAD6, a specific monoclonal antibody to GAD65. The MGAD·anti-GAD immunoprecipitate was subjected to SDS-PAGE and immunoblot analysis with anti-HSP70 polyclonal antibodies. HSC70 was found to associate with GAD·anti-GAD65 complex in the immunoprecipitate (lane C) as indicated by anti-HSP70 staining, whereas no detectable HSC70 was observed in the supernatant after immunoprecipitation (lane B).

Presence of GAD₆₅, GAD₆₇, HSC70, and CSP on Synaptic Vesicles-- To determine whether GAD₆₅, GAD₆₇, HSC70, and CSP are present on synaptic vesicles, highly purified synaptic vesicle preparations devoid of soluble proteins (15) were probed with various antibodies on immunoblots. As shown in Fig. 5 (lane 1), a protein band at the position of CSP is strongly stained with anti-CSP polyclonal antibodies, although a few other protein bands are also visible. Anti-HSP70 identified a single protein band at the position corresponding to a molecular mass at about 67 kDa (Fig. 5, lane 2). Anti-GAD₆₅ and anti-GAD₆₇ preadsorbed by respective antigens recognized single bands representing specific isoforms of GAD₆₅ and GAD₆₇ (Fig. 5, lanes 3 and 4), whereas the non-preadsorbed anti-GAD₆₅ and GAD₆₇ polyclonal antibodies revealed both bands as well as an occasional lower band at a molecular mass range of 60-67 kDa (results not shown). The identity of GAD₆₅ was further confirmed by GAD6, which stained a protein band (Fig. 5, lane 5) at the same position as indicated by the preadsorbed GAD₆₅ antibodies (Fig. 5, lane 3). These results clearly indicate the presence or association of GAD₆₅, GAD₆₇, HSC70, and CSP with synaptic vesicles.

View larger version (78K): [in this window] [in a new window]   Fig. 5.   Immunoblot detection of GAD65, GAD67, HSP70, and CSP on highly purified synaptic vesicles. Highly purified synaptic vesicle preparations obtained by density gradient centrifugation and size exclusion chromotography columns were separated by SDS-PAGE and analyzed by immunoblotting. Immunoblots were probed with the following antibodies: lane 1, anti-CSP; lane 2, anti-HSP70; lane 3, anti-GAD65; lane 4, anti-GAD67; lane 5, GAD6.

Association of Bacterial Homologue of HSC70, DnaK, with Recombinant GAD₆₅ and GAD₆₇-- Since the E. coli system in general does not share similar posttranslational modifications with mammalian cells, it would be of interest to see whether recombinant HGAD₆₅ and HGAD₆₇ can also form protein complexes with the bacterial homologue of HSC70, DnaK (19). As shown in Fig. 6, both GST-HGAD₆₅ and GST-HGAD₆₇ fusion proteins purified by GSH affinity columns (8) contain GAD and DnaK as indicated by positive identification in immunoblotting tests using specific anti-HSP70 and anti-GAD sera. Because the difference of the molecular masses between GAD₆₅, GAD₆₇, and HSC70 is small, the larger proteins, namely GST-GAD₆₅ and GST-GAD₆₇ fusion proteins (91-93 kDa), were specifically used in immunoblots to show clear separation of these proteins and the lack of cross-reactivity between anti-HSC70 antibodies and GST-GADs. As shown in Fig. 6, no immunostaining was observed by anti-HSP70 at the gel position corresponding to GST-GAD₆₇ (Fig. 6, lane 2) and GST-GAD₆₅ (Fig. 6, lane 4), thus indicating the lack of cross-reactivity between either anti-HSP70 and GST-GAD₆₇ or GST-GAD₆₅.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Immunoblots of partially purified recombinant GST-GAD65, GAD67 fusion proteins with anti-GAD and anti-HSP70. Partially purified isoforms of recombinant GST-human GAD, GST-HGAD65, and GST-HGAD67, fusion proteins were subjected to SDS-PAGE and immunoblot analysis with anti-GAD65 (lane 1), and anti-GAD67 (lane 3). The same polyvinylidene difluoride blots were stripped and reprobed with anti-HSP70 (lanes 2 and 4). The arrows indicate the separate positions of the HSC70 bacterial homolog DnaK and the isoforms of GST-GAD fusion proteins.

Demonstration of Protein Complex of GAD, HSC70, and CSP in Immunoaffinity-purified GAD Preparations-- Protein complexes of GAD, HSC70, and CSP were isolated from crude brain subcellular extracts of S1 and P₂M using anti-GAD₆₅ and anti-GAD₆₇ immunoaffinity columns. The conditions used were of high stringency to prevent nonspecific protein binding during the affinity column purification. The columns were washed extensively with high salt buffer solution after application of the brain extract. The results showed that the GAD preparations purified by anti-GAD immunoaffinity columns also contain HSC70 and CSP in addition to GAD (Fig. 7). This suggest that HSC70 and CSP form a protein complex with GAD and hence were co-purified by the anti-GAD immunoaffinity columns. Retention of HSC70 was observed in all S1 and P₂M fractions, suggesting that GAD·HSC70 complex is soluble as well as membrane-anchored. Slight retention of GAD₆₅ in the anti-GAD₆₇ columns and GAD₆₇ in the anti-GAD₆₅ columns was observed in both S1 and P₂M fractions as indicated by immunoblotting tests.² These results showed that both GAD₆₅ and GAD₆₇ are present as soluble as well as the membrane-bound forms. Interestingly, quantitative analysis of these immunoblots indicated that GAD₆₅ protein is roughly distributed equally between the soluble and membrane fractions, whereas GAD₆₇ protein is present largely (~80%) as soluble form. Although the crucial amino acid sequences involved in membrane anchoring of GAD₆₅ have been determined (20), the nature of membrane association of GAD₆₇ remains largely uncharacterized. One hypothesis is that the membrane association characteristic of GAD₆₇ is due to the heterodimer formation of GAD₆₇ to the membrane-associated GAD₆₅ (21, 22). Judging by the intensity of the CSP band on immunoblots, most CSP retained by the affinity columns is present in the membrane fractions (Fig. 7, lanes 2 and 4) with slight retention of CSP in the S1 fractions by anti-GAD₆₅ affinity column (Fig. 7, lane 1). It is unlikely that anti-GAD₆₅ and anti-GAD₆₇ may cross-react with HSC70 or CSP, since the results shown in Figs. 5 and 6 clearly indicate the lack of immunostaining between anti-GAD₆₅ as well as anti-GAD₆₇ with either HSC70 or CSP. Therefore, the retention of HSC70 (Fig. 7, lanes 5-8) and CSP by anti-GAD immunoaffinity columns is the result of protein-protein interaction between GAD, HSC70, and CSP.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Immunoblot analysis of immunoaffinity purified GAD complexes with anti-HSP70 and anti-CSP. GAD preparations obtained from porcine brain extracts, S1 and P2M, were purified through either an anti-GAD65 (lanes 1, 2, 5, and 6) or an anti-GAD67 column (lanes 3, 4, 7, and 8). The eluate were subjected to SDS-PAGE and immunoblot analysis, with anti-HSP70 (lanes 5-8) and anti-CSP (lanes 1-4). The upper and lower arrows indicate the position of HSC70 and CSP, respectively.

Activation of GAD Activity by HSC70 and Highly Purified Synaptic Vesicles-- To assess the effect of protein-protein interaction on GAD activity, reconstitution experiments were conducted using purified HGAD₆₅, purified HSC70, and highly purified synaptic vesicle preparations, which have been shown to retain electrochemical proton gradient (23, 24). Since synaptic vesicles and HSC70 alone do not contain detectable GAD activity (Fig. 8, lanes 1 and 2), it is interesting to note that GAD₆₅ activity is markedly increased by incubation with either HSC70 (Fig. 8, lane 6), or synaptic vesicles (Fig. 8, lane 5), as well as HSC70 together with synaptic vesicles (Fig. 8, lane 7). Bovine serum albumin, which was included as control protein, showed no effect on GAD activity (Fig. 8, lane 4). These observations are compatible with our hypothesis that GAD₆₅ is anchored to synaptic vesicles through HSC70 and its activity is increased through an electrochemical proton gradient-mediated process (5). Furthermore, the formation of protein complex between GAD₆₅ and HSC70 as indicated by co-purification (Figs. 2 and 7), reconstitution assay (Fig. 3), and co-immunoprecipitation (Fig. 4) may induce conformational changes on the GAD₆₅ favorable to its stability and catalytic activity.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Activation of GAD65 by protein complex formation with HSC70 and synaptic vesicle. A highly purified isoform of human recombinant GAD, HGAD65 (10 µg), was incubated with purified biotinylated bovine HSC70 (3 µg) and/or highly purified synaptic vesicle (SV) (5 µg) at room temperature for 1 h. The incubation mixtures were then assayed for GAD activity as described previously (5). Lane 1, SV alone; lane 2, HSC70 alone; lane 3, GAD65 alone; lane 4, GAD65 + bovine serum albumin; lane 5, GAD65 + SV; Lane 6, GAD65 + HSC70; lane 7, GAD65 + SV + HSC70. Activation of GAD activity was observed by incubations with HSC70, SV, or a combination of both but not with bovine serum albumin. HSC70 and SV alone do not display GAD activity. Error bars indicate standard deviations with n = 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Since the structures of various isoforms of GAD have been determined (for review, see Ref. 2), it becomes clear that, in general, GAD is not an integral membrane protein due to lack of a stretch of hydrophobic amino acids long enough to span the membrane. Nevertheless, some populations of both GAD₆₅ and GAD₆₇ are still firmly anchored to membranes despite various ionic extraction methods (20-22). GAD can interact with membranes by ionic or hydrophobic mechanisms. Fonnum (25) reported that GAD could become associated with membrane in the presence of Ca²⁺. Martin and Martin (26) have shown that apoGAD has a strong affinity for polyanions, e.g. hexasulfate, and thus may be anchored to synaptic vesicles through ionic interactions, since the cytoplasmic face of synaptic vesicles is enriched in acidic phospholipids (27). However, Chang and Gottlieb (18) have demonstrated that 60% of GAD is membrane bound and can be released only with detergent (e.g. 0.2% Triton X-100) but not with high salt, e.g. 1 M NaCl or 1 M KCl, suggesting that the interaction between GAD and membranes is predominantly hydrophobic. Subsequent studies attempting to characterize this hydrophobic interaction included studies in palmitoylation as a membrane anchorage mechanism of GAD₆₅ (28). However, a more recent study showed that palmitoylation is not required for anchoring GAD₆₅ to membranes since mutation of amino acids involved in palmitoylation has no effect on membrane anchorage (20). Recent efforts in elucidating the membrane association mechanism of GAD by protein phosphorylation (29) and GAD dimerization (21, 22) have deciphered the respective crucial amino acid sequences involved. Still, the mechanism of GAD anchorage to membranes and synaptic vesicles and its effect on GAD activity remains unclear.

Besides a direct interaction with membranes as discussed above, proteins can also become membrane-anchored through chaperone proteins. One example is the 70-kDa family of heat shock proteins (HSP70), which have been shown to serve as molecular chaperons in promoting protein folding and facilitating protein transport to intracellular organelles, including mitochondria, nuclei, endoplasmic reticulum, and lysosomes (30-32). The ubiquitous binding partners of HSP70 include a wide range of unfolded peptides as well as native proteins. Two native proteins found in the nerve terminal that bind with the constitutively expressed member of the heat shock protein 70 family, HSC70, are the clathrin triskelions of coated vesicles (33, 34) and the intrinsic synaptic vesicle protein, CSP (35-37).

In this study, several lines of evidence are presented to support the hypothesis that GAD can become membrane-associated or anchored to synaptic vesicles through protein complex formation first with HSC70, followed by protein-protein interaction involving GAD·HSC70 complex and CSP on synaptic vesicles. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form a complex with GAD₆₅ as shown in gel shift mobility in NG- PAGE. Third, in immunoprecipitation studies, again, HSC70 was found to co-immunoprecipitate with GAD by a GAD₆₅-specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. In light of our previous findings that MGAD is activated by protein phosphorylation, which depends on the integrity of the proton gradient on synaptic vesicles and is inhibited by dephosphorylation (5), whereas SGAD is activated by dephosphorylation and inhibited by phosphorylation (6, 7), the physiological significance of GAD association to synaptic vesicles and its activation by interaction with HSC70 and synaptic vesicles can be summarized in Fig. 9. Once GABAergic neurons are stimulated, GABA is released by exocytosis (). Synaptic vesicles are then retrieved through coated vesicle formation. Clathrin-coated pits are then dissociated from vesicles through interaction with HSC70 (). The uncoated synaptic vesicles thus replenish their proton gradient through action of the vacuolar proton pump (V-ATPase) (). Once the vesicular proton gradient is restored, MGAD, which is anchored to synaptic vesicles through HSC70 and CSP is activated by membrane-bound protein kinase (). Newly synthesized GABA is then transported into synaptic vesicles by the vesicular GABA transporter to replenish vesicular GABA (). In addition, activation of the GABA neurons triggers the influx of Ca²⁺ into the terminals (), which activates calcineurin (PrP 2B) (), and results in increased GABA synthesis by SGAD. GABA synthesized by SGAD may also be transported into synaptic vesicles () or be catabolized by GABA-transaminase to provide ATP (), which may be utilized by V-ATPase to maintain the proton gradient on synaptic vesicles, a condition favoring GABA synthesis by MGAD and GABA transport by vesicular GABA transporter.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   A proposed model on the anchorage of GAD65 to synaptic vesicles at the synaptic terminal. GAD65 is subcellularly targeted and anchored to synaptic vesicles first through the chaperone function of HSC70 to form a HSC70·GAD65 complex, followed by association of HSC70·GAD65 complex to CSP on synaptic vesicles. See "Discussion" for details.

	FOOTNOTES

^* This work was supported in part by National Science Foundation Grant IBN-9723079, Office of Naval Research Grant N00014-94-1-04572, the J. R. & Inez W. Jay Biomedical Research Fund, and the Research Development Fund (University of Kansas).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence and reprint requests should be addressed. Tel.: 785-864-4557; Fax: 785-864-5374; E-mail: jywu@ukans.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M001403200

² C.-C. Hsu, K. M. Davis, H. Jin, T. Foos, E. Floor, W. Chen, J. B. Tyburski, C.-Y. Yang, J. V. Schloss, and J.-Y. Wu, unpublished results.

	ABBREVIATIONS

The abbreviations used are: GAD, L-glutamate decarboxylase; GABA, -aminobutyric acid; SGAD, soluble L-glutamate decarboxylase; MGAD, membrane-associated L-glutamate decarboxylase; HGAD, human L-glutamate decarboxylase; CSP, cysteine string protein; HSC, heat shock cognate; PLP, pyridoxal 5'-phosphate; AET, 2-aminoethylisothiuronium bromide; NG, non-denaturing gradient; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; AMP-PNP, adenosine 5'-(,-imino)triphosphate; SV, synaptic vesicle.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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