Dissociation of αGDI from the Hsp90 chaperone complex is dependent on Ca²⁺-induced neurotransmitter release

To begin to address the physiological role of the Hsp90-containing chaperone complex in αGDI activity and Rab recycling, we examined the relationship between complex formation and Ca²⁺-induced neurotransmitter release in DPS. Like streptolysin-permeabilized synaptosomes (Tandon et al., 1998), incubation of washed DPS in vitro resulted in the efficient (>50%) release of the neurotransmitter glutamate in a reaction that was dependent on the addition of exogenous Ca²⁺ and required ATP, as apyrase potently inhibited release (Figure 2A). Furthermore, release required GTP, as the presence of the non-hydrolyzable analog GTPγS (1 mM) inhibited Ca²⁺-dependent glutamate release from DPS by ~40% compared with the control (Figure 2A). We also examined whether Ca²⁺-dependent transmitter release required SNAp-REceptors (SNAREs) involved in vesicle fusion (Sollner et al., 1993). Glutamate release was sensitive to N-ethylmaleimide (NEM), an inhibitor of SNARE function (Matveeva and Whiteheart, 1998) (Figure 2B). Moreover, when DPS were pretreated with recombinant light chain of either botulinum (Bot) toxin B or E at 30°C for 15 min, washed and assayed for glutamate release stimulated by Ca²⁺, the Bot B toxin inhibited glutamate release >80%, whereas Bot E inhibited release by ~50%, similar to values reported previously for streptolysin-permeabilized synaptosomes (Tandon et al., 1998) (Figure 2C).

Open in a separate window

Fig. 2. Dissociation of αGDI from the Hsp90 chaperone complex is dependent on Ca²⁺-induced neurotransmitter release. (A–C) Requirement for energy and SNARE function for Ca²⁺-mediated glutamate release from DPS as measured spectrofluorometrically. (A) DPS were incubated in the absence or presence of the ATP-regenerating system (ATP mix), or in the presence of GTPγS or treated with 20 U of apyrase for 15 min at 30°C, washed and assayed for glutamate release in the absence of the ATP mix. (B) Where indicated, synaptosomes were pretreated with NEM before permeabilization. (C) DPS were treated with recombinant light chains of Bot B and E toxins for 15 min at 30°C, and washed prior to Ca²⁺-stimulated glutamate release. (D) Effects of Bot B and E on dissociation of αGDI from the Hsp90-containing chaperone complex. Untreated (lanes a and b), Bot B-treated (lanes c and d) or Bot E-treated DPS (lanes e and f) were incubated in the absence (a, c and e) or presence (b, d and f) of 10 µM Ca²⁺ and incubated for 15 min at 30°C. After Ca²⁺ stimulation, membranes were solubilized and αGDI-containing complexes were immunoprecipitated using the anti-αGDI mouse monoclonal antibody (left panel) and quantitated (right panel) by immunoblotting. Results shown in all panels are representative of at least three independent experiments.

To examine whether the assembly of the αGDI–Hsp90 complex was sensitive to Ca²⁺-induced neurotransmitter release, synaptosomes were incubated at 30°C for 15 min in the absence or presence of Ca²⁺, DPS were treated with the cleavable cross-linker, solubilized with lysis buffer and immunoprecipitated with αGDI-specific antibody. In the presence of Ca²⁺, we observed an ~70% dissociation of Hsp90 from the αGDI–chaperone complex and an ~50% reduction in the amount of Hsc70 and CSP recovered in the αGDI immunoprecipitate (Figure 2D, lanes a and b). The latter result was confirmed by immunoprecipitation with CSP-specific antibody (data not shown). While treatment with Bot E had only a small effect on complex dissociation (Figure 2D, lanes e and f), consistent with its modest effect on transmitter release, pretreatment with Bot B reduced the ability of αGDI to dissociate from the chaperone complex ~30% compared with controls (Figure 2D, lanes c and d). These results suggest that Ca²⁺ stimulation triggers the dissociation of αGDI from the chaperone complex, a condition that favors Rab3A GTP hydrolysis, synaptic vesicle fusion and recycling of Rab3A by αGDI. Thus, function of the SNARE fusion machinery correlates with the ability of the αGDI– chaperone complex to disassemble.

The Hsp90 inhibitors GA and radicicol block both neurotransmitter release and αGDI release from the chaperone complex

To further explore the role of Hsp90 in the αGDI/Rab3A cycle, we examined the effect of the Hsp90 inhibitor GA on Ca²⁺-induced neurotransmitter release using intact synaptosomes and DPS. GA has previously been shown to bind to the ATP site of Hsp90 and prevent interaction with its co-chaperone partners, thereby preventing activation of downstream signaling pathways (Richter and Buchner, 2001; Young et al., 2001). While pretreatment of synaptosomes with GA for 10 min at 30°C had no effect on their morphology, based on electron microscopy, and no effect on the total content of glutamate in synaptosomes (data not shown), it had a potent effect on Ca²⁺-induced neurotransmitter release. As shown in Figure 3, neurotransmitter release was blocked in a dose-dependent fashion from both intact synaptosomes (Figure 3A) and DPS (Figure 3B). As expected, inhibition could be competed by the addition of high levels of ATP (Figure 3C). Using morphometry (Supplementary figure 2), we observed a 40–60% reduction in the size of the synaptic vesicle pool in DPS in response to Ca²⁺, a result that was nearly completely inhibited in the presence of GA. To provide additional evidence that Hsp90 is involved in αGDI function, we used a structurally and chemically different Hsp90 inhibitor, the macrocyclic drug radicicol, known to bind to Hsp90 with high specificity and to inhibit its ATPase activity (Roe et al., 1999). We found that a 10 min pretreatment of synaptosomes with radicicol inhibited neurotransmitter release in a dose-dependent manner from both intact synaptosomes (data not shown) and DPS (Figure 3D).

Open in a separate window

Fig. 3. Dissociation of αGDI from the Hsp90-containing chaperone complex is prevented by GA and radicicol. (A–D) Sensitivity of glutamate release to Hsp90 inhibitors. (A) Intact synaptosomes were pretreated with GA, SB 203580 (SB) or DMSO (as a control) and then depolarized by the addition of KCl (30 mM). (B) Intact synaptosomes were pretreated with GA, SB or DMSO before permeabilization and then stimulated for glutamate release by the addition of Ca²⁺ (10 µM). (C) Intact synaptosomes were pretreated with GA or DMSO before permeabilization and in the absence or presence of an ATP-regenerating system (ATP mix) and stimulated for glutamate release by the addition of Ca²⁺. (D) Intact synaptosomes were pretreated with radicicol (Rad) or DMSO before permeabilization and then stimulated for glutamate release by the addition of Ca²⁺. (E) Effect of GA and radicicol on dissociation of αGDI from the Hsp90-containing chaperone complex. Untreated (lanes a and b), GA-treated (lanes c and d) or radicicol-treated DPS (lanes e and f) were incubated in the absence (a, c and e) or presence (b, d and f) of 10 µM Ca²⁺ and incubated for 15 min at 30°C. After stimulation, membranes were solubilized and αGDI-containing complexes were immunoprecipitated using the anti-αGDI mouse monoclonal antibody (left panel) and quantitated (right panel) by immunoblotting. The results shown for all panels are representative of at least three independent experiments.

To examine whether the effects of Hsp90 inhibitors on neurotransmitter release were associated with changes in the binding of αGDI with the chaperone complex, we examined the ability of αGDI to remain bound to Hsp90, Hsc70 and CSP in response to Ca²⁺-induced neurotransmitter release in the absence or presence of GA or radicicol. Both GA and radicicol significantly reduced the ability of αGDI to cycle through the chaperone system (Figure 3E), preventing normal αGDI function. These results suggest that Ca²⁺ may activate release of αGDI from the Hsp90-containing complex in a step coordinated with Rab3A recycling and neurotransmitter release.

Formation of the αGDI–Hsp90 complex is ATP dependent and requires the function of the αGDI mobile effector loop (MEL)

The assembly of steroid hormone receptor–Hsp90 complexes and activation of steroid binding activity by purified chaperones require ATP hydrolysis (Richter and Buchner, 2001; Young et al., 2001). Thus, we wanted to determine whether the assembly of αGDI with Hsp90, Hsc70 and CSP requires ATP. For this purpose, we attached recombinant His₆-tagged αGDI protein to protein A– Sepharose beads (αGDI–beads). αGDI–beads were incubated with purified Hsp90, Hsc70 and recombinant CSP in the presence of an ATP-regenerating system. As shown in Figure 4A, αGDI incubated with Hsp90, Hsc70 and CSP in the presence of an ATP-regenerating system formed an αGDI–Hsp90 heterocomplex. In contrast, in the presence of the non-hydrolyzable analog AMP-PNP, heterocomplex formation was blocked. Given the ability of GA to interfere with normal αGDI function in DPS, the dose–response effect of GA on complex assembly was examined by SDS–PAGE followed by Coomassie Blue staining (Figure 4B). In a dose-dependent manner, GA reduced recovery of Hsp90 in a αGDI-containing complex by ~50% and increased the recovery of Hsc70. Quantitative densitometry of stained protein bands suggested half-maximal inhibition of association with the complete complex at ~7.5 µM GA. Thus, assembly in vitro of αGDI with Hsp90 requires the normal assembly pathway observed for steroid hormone receptors that may first involve recruitment to Hsc70/Hsp40 components (Richter and Buchner, 2001; Young et al., 2001). As Ca²⁺ did not affect the assembly of the complex in vitro (Figure 4B), we suggest that additional factors facilitate Ca²⁺-stimulated association events observed in DPS.

Open in a separate window

Fig. 4. Formation of αGDI–Hsp90 complexes is ATP dependent and requires the function of the αGDI MEL. (A) Requirement for MEL. Lanes a, d, g and j, His₆-αGDI–beads (S) incubated with ATP-regenerating in buffer alone; lanes b, e, h and k, His₆-αGDI–beads incubated with purified Hsp90, Hsc70 and recombinant CSP in the presence of the ATP-regenerating system; lanes c, f, i and l, His₆-αGDI–beads incubated with purified Hsp90, Hsc70 and CSP in the presence of the ATP-regenerating system containing AMP-PNP instead of ATP. Bound proteins were separated by SDS–PAGE, stained with Coomassie Blue (upper panel) and quantitated (lower panel) by densitometry. (B) Dose-dependent effect of GA on the formation of the Hsp90–αGDI–chaperone complex. His₆-αGDI–beads containing wild-type αGDI were incubated in the presence of the ATP-regenerating system with DMSO-treated chaperones (lane a), with the indicated concentration of GA (lanes b–d) treated chaperones (lane e) in the presence of Ca²⁺. Proteins used for reconstitution are indicated in the left three lanes. Bound proteins were separated by SDS–PAGE, stained with Coomassie Blue (upper panel) and quantitated (lower panel). The results shown in both panels are representative of at least three independent experiments.

To provide additional evidence for the specificity of the in vitro assembly reaction, we have previously demonstrated the importance of the Rab-binding domain (RBD) in domain I of αGDI for interaction with Rab (Schalk et al., 1996; Wu et al., 1998; Luan et al., 1999), and the domain II MEL for Rab recycling and interaction with RRF (Luan et al., 2000). To address the importance of Rab-binding and MEL function in αGDI–Hsp90 complex assembly in vitro, we generated αGDI mutants that inhibit separately the function of the key residues in the RBD (αGDI[R240A]) (Schalk et al., 1996; Wu et al., 1998; Luan et al., 1999) and MEL (αGDI[R218E]) (Luan et al., 2000), as well as a double mutant inhibiting the function of both domains (αGDI[R218E/R240A]). These αGDI mutants were bound to beads and incubated with Hsp90, Hsc70 and CSP in the presence of the ATP-regenerating system. Strikingly, we found that αGDI[R218E] and αGDI[R218E/R240A] mutants did not recruit Hsp90 and showed a marked reduction in binding of Hsc70 and CSP (Figure 4A). In contrast, wild-type αGDI and the αGDI[R240A] mutant showed equivalent interaction with Hsp90, Hsc70 and CSP. These results suggested that MEL is important for the specific association with the Hsp90 complex.

GA inhibits Rab3A extraction by endogenous αGDI

To determine whether the effects of GA directly affect the ability of endogenous αGDI to extract Rab3A from membranes during vesicle fusion, we examined the redistribution of Rab3A and αGDI in DPS in response to Ca²⁺ stimulation. In the absence of GA, we found that αGDI could efficiently retrieve Rab3A from the membrane in response to Ca²⁺-induced neurotransmitter release (Figure 5, lanes a–d). The stoichiometry of the recovered αGDI–Rab3A cytosolic complex was 1:1 based on quantitative immunoblotting (data not shown). In contrast, we found that GA potently inhibited Rab3A retrieval from membranes in response to Ca²⁺-induced neurotransmitter release (Figure 5, lanes e–h). These results suggest that GA may inhibit Ca²⁺-induced neurotransmitter release in response to a defect in the retrieval of Rab3A from membranes through the activity of an intermediate Hsp90-containing chaperone complex.

Open in a separate window

Fig. 5. GA inhibits Ca²⁺-induced Rab3A recycling. DPS preincubated in the absence (lanes a–d) or presence (lanes e–h) of GA (5 µM) were incubated for 15 min at 30°C in the absence (a, b, e and f) or presence (c, d, g and h) of Ca²⁺ (10 µM). Following incubation, membranes were collected by centrifugation, and the resulting soluble fraction was immunoprecipitated with anti-αGDI. The immunoprecipitate of the soluble fraction (S) (50% of total shown) and the solubilized membrane pellet fraction (P) (10% of total shown) (upper panel) were quantitated by immunoblotting with anti-αGDI and Rab3A antibodies as indicated (lower panel). The results shown are representative of at least three independent experiments.

A chaperone complex containing Hsp90 and CSP modulates αGDI function

We have provided four independent lines of evidence that Hsp90 is likely to play a critical role in the αGDI cycle of Rab3A in the synapse: (i) co-immunoprecipitation of endogenous αGDI with Hsp90; (ii) the ability of GA/radicicol, inhibitors of Hsp90 chaperone complex function (Richter and Buchner, 2001; Young et al., 2001), to prevent recycling of Rab3A by endogenous αGDI; (iii) the ability of GA/radicicol to prevent the ability of exogenous GST–αGDI to retrieve Rab3A from synaptic membranes under physiological conditions; and (iv) the specific role of MEL to facilitate direct interaction of αGDI with the Hsp90-containing complex. Moreover, we found substantial correlative results between the ability of GA/radicicol to disrupt Rab3A recycling, interfere with αGDI association with the Hsp90 complex and inhibit Ca²⁺-dependent neurotransmitter release.

In order to determine the molecular partners involved in αGDI function in Rab3A recycling, we utilized synaptosomes permeabilized with digitonin. In contrast to the variable activity of different preparations of streptolysin used to perforate synaptosomes in previous studies (Tandon et al., 1998), we have found that the digitonin permeabilization technique described herein is highly reproducible and allows the measurement of neurotransmitter release in vitro in real time with high efficiency. This technique should provide a useful new tool to study the role of other components mediating neurotransmitter release.

Using DPS, we identified a membrane-associated detergent soluble pool that, in the absence of cross-linker, showed efficient binding of αGDI to Hsp90. This interaction was strengthened in the presence of cross-linker to a near stoichiometric complex. Moreover, in the presence of cross-linker, we recovered Hsc70 and CSP, a synaptic vesicle-associated DnaJ domain-containing protein (Chamberlain and Burgoyne, 2000), as well as additional proteins of unknown identity. These results suggest that a major component of a membrane-associated complex containing αGDI is likely to include Hsp90 tethered to synaptic vesicles, through CSP/Hsc70 and other factors. The potential association of the αGDI with the Hsp90 chaperone complex and with synaptic vesicles was supported by subfractionation, and by biochemical studies in which these interactions were demonstrated using purified, recombinant proteins. This reaction specifically involved MEL, a key effector region in domain II of αGDI required for Rab retrieval through the RRF (Luan et al., 2000).

Hsp90 complexes are now recognized to contain a number of facilitating co-chaperones that modulate function (Buchner, 1999). The potential importance of both Hsc70 and CSP in a vesicle targeting/fusion complex is supported by separate studies in which CSP (Ranjan et al., 1998) and Hsc70 (Bronk et al., 2001) have been suggested to play important roles in exocytosis and modulation of Ca²⁺ sensitivity. In contrast, we have been unable to detect the co-chaperones Hop and Hip involved in steroid hormone signaling (Richter and Buchner, 2001; Young et al., 2001; T.Sakisaka and W.E.Balch, unpublished observations). These proteins contain tetratricopeptide repeat-containing domains (TPRs), a motif that interacts with Hsp90 and Hsp/Hsc70. The lack of Hop or Hip is consistent with the fact that TPR-containing proteins are generally specific for a particular Hsp90-regulated signaling pathway (Blatch and Lassle, 1999). Interestingly, CSP and Hsc70 have recently been shown to form a molecular complex of unknown function on synaptic vesicles that binds the TPR-containing protein SGT, a CSP-interacting protein that has been proposed to function as a co-chaperone like Hip (Hohfeld et al., 1995; Tobaben et al., 2001). Thus, there remains the possibility currently under investigation that SGT and/or other unknown proteins are part of the αGDI–Hsp90 chaperone complex that facilitates Rab3A recycling.

Regulation of Rab3A recycling and neurotransmitter release in response to GA

While the identification of a chaperone complex containing αGDI represents an important step forward in defining novel binding partners participating in membrane association and Rab recycling, we have provided evidence that this complex may be physiologically important during neurotransmitter release. For this purpose, we made use of both GA and radicicol, specific inhibitors of Hsp90 function. GA/radicicol have been demonstrated to tightly bind to the ATP site of Hsp90. Binding of GA/radicicol is currently thought to prevent assembly of the Hsp90-containing complexes from intermediate Hsc70/Hsp40-containing complexes and thereby block the activity of Hsp90-regulated signaling pathways (Whitesell et al., 1994; Prodromou et al., 1997; Stebbins et al., 1997). We provided evidence that both drugs inhibited αGDI activity in Rab3A extraction. Thus, the effect of GA/adicicol on Rab3A recycling may be to inhibit the Hsp90 chaperone-mediated function of the client molecule αGDI.

Of particular interest is the fact that GA is also known to be an inhibitor of a number of kinase-regulated cascades through its effect on Hsp90 co-chaperone activity (Richter and Buchner, 2001; Young et al., 2001). Recently, Gruenberg and colleagues (Cavalli et al., 2001) have demonstrated that the MAP kinase p38, a serine protein kinase sensitive to the inhibitor SB203580, is involved in the regulation of GDI function in recycling of the Rab5 GTPase directing endosomal trafficking (Cavalli et al., 2001). p38 can phosphorylate Ser121 of αGDI, a residue adjacent to the MEL region that directs interaction with RRF (Luan et al., 2000). In contrast to the potential role of p38 in Rab5 recycling, we found no effect of SB203580 on the ability of αGDI to recycle Rab3A in synaptosomes, suggesting that the pathways regulating GDI function may differ for different Rab GTPases.

In addition to the effects of GA/adicicol on αGDI-mediated Rab3A recycling, a correlation was observed between the effects of these drugs on inhibition of Rab3A retrieval from membranes and their ability to inhibit Ca²⁺-dependent glutamate release. Disruption of MEL reduced the ability of wild-type αGDI to promote Rab3A recycling, interact with Hsp90 and interfere with neurotransmitter release in vitro. Consistent with these effects on intact and DPS, we have also observed a partial effect of GA on neurotransmitter release from culture hippocampal neurons (C.Stevens and W.E.Balch, unpublished observations). While further work is necessary to elucidate the basis for these results, one possibility is that Hsp90 facilitates an unknown step in addition to the αGDI-mediated Rab3A recycling, triggering vesicle targeting/fusion. Alternatively, and consistent with our evidence, a second possibility is that by interfering with the cycling of αGDI through the Hsp90-containing chaperone complex, a Rab3A-containing priming complex (Castillo et al., 2002; Schoch et al., 2002) is unable to efficiently mature in response to Rab3A hydrolysis. Thus, the presence of Rab-GDP may have an unsuspected effect on protein interactions dictating downstream targeting/fusion events related to synaptic plasticity (long-term potentiation; LTP). Our results are consistent with the current view that Rab3A regulates the availability of synaptic vesicles for membrane fusion (Geppert and Sudhof, 1998).

Preparation of permeabilized synaptosomes

Intact synaptosomes were suspended at a protein concentration of 1 mg/ml in buffer A [50 mM HEPES–KOH pH 7.2, 100 mM KCl, 5 mM MgCl₂, 2 mM EGTA, 1 mM DTT and complete protease inhibitor cocktail (Sigma)] and incubated with 50 µg/ml digitonin for 10 min on ice. Digitonin-treated synaptosomes were washed with buffer A and resuspended in buffer B (50 mM HEPES–KOH pH 7.2, 40 mM KCl, 5 mM MgCl₂, 2 mM EGTA, 1 mM DTT and complete protease inhibitor cocktail) for 10 min at 4°C to allow for washout of soluble proteins.

Cross-linking of synaptosome proteins

Intact synaptosomes were resuspended at a protein concentration of 1 mg/ml in buffer C (20 mM HEPES–NaOH pH 7.4, 140 mM NaCl, 5 mM KCl, 5 mM NaHCO₃, 1.2 mM Na₂HPO₄, 1 mM MgCl₂ and 10 mM glucose) and pre-warmed for 5 min at 30°C. Cross-linker (dissolved in DMSO) was then added to yield a final concentration of 1 mM (1% v/v of the solvent). After incubation with stirring for 15 min at 30°C, 50 mM Tris–HCl at pH 7.4 was added as a quencher, followed by osmotic shock. Synaptosomes were subsequently fractionated by differential centrifugation as described previously (Stahl et al., 1996) and αGDI-interacting proteins recovered by immunoprecipitation.

Solubilization and isolation of αGDI complexes from synaptosome membranes

DPS were prepared as described in Supplementary figure 2, resuspended in buffer A and solubilized with 1% Triton X-100 for 1 h. Insoluble material was removed by centrifugation, the supernatant diluted with two volumes of buffer A and the diluted sample (1 ml containing 1 mg of protein) immunoprecipitated by the addition of 15 µg of the anti-αGDI covalently coupled to 50 µl of protein A–Sepharose 4B beads (Pharmacia). A mouse immunoglobulin G was used as a non-specific control. For CSP immunoprecipitation, the detergent extract was incubated with 20 µg of an anti-CSP rabbit polyclonal antibody covalently coupled to 50 µl of protein A–Sepharose 4B beads.

Glutamate release assay

The glutamate release assay was performed using enzyme-linked fluorescent detection of released glutamate (Nicholls and Sihra, 1986; Supplementary figure 2).

Release of Rab3A from DPS

DPS were resuspended with buffer B (1 mg of protein per 1.5 ml of buffer). After incubating with stirring for 5 min at 30°C, DPS were stimulated with 10 µM CaCl₂, incubated for an additional 15 min, and membranes were collected by centrifugation. The supernatant was incubated with the anti-αGDI mouse monoclonal antibody. Fifty percent of the immunoprecipitate and 50 µl of the membrane pellet were resuspended in a total of 500 µl of SDS sample buffer, separated using SDS–PAGE and immunoblotted with anti-αGDI and anti-Rab3A rabbit polyclonal antibodies.

Retrieval of Rab3A by GST–αGDI and His₆-αGDI

Twenty-five micrograms of SM and the indicated concentration of GST–αGDI (see Results) were supplemented with 250 µM GDP in a 200 µl final volume of buffer A. After incubation for 20 min at 37°C, the samples were chilled on ice and membranes pelleted by centrifugation for 20 min at 250 000 g. The membrane pellet was resuspended in a total of 80 µl of SDS sample buffer. The supernatant was incubated with 50 µl of glutathione–Sepharose (GS) beads for 1 h at 4°C. Fifty percent of GS–bead recovered αGDI and the 40 µl of the solubilized membrane pellet were analyzed using SDS–PAGE and immunoblotting with anti-αGDI and Rab3A rabbit polyclonal antibodies. Where indicated in the Results, the SM were preincubated with either GA, SB 203580 and DMSO (control) for 10 min at 30°C prior to incubation with GST–αGDI. His₆-αGDI was incubated with cobalt beads (TALON metal affinity beads; Clontech) to recover the His₆-αGDI–Rab3A complex.

Binding of wild-type and mutant His₆-αGDI to Rab3A

Twenty-five micrograms of 0.6% CHAPS-extracted SM (diluted below the critical micelle concentration at 0.18%) and the indicated concentration of His₆-αGDI (see Results) were supplemented with 250 µM GDP in a 200 µl final volume of buffer A supplemented with CHAPS (final concentration 0.18%). After incubation for 20 min at 37°C, the samples were chilled on ice, centrifuged for 20 min at 250 000 g, and the supernatant incubated with 50 µl of cobalt beads for 1 h at 4°C. Cobalt bead-recovered His₆-αGDI (50% of total) was analyzed by SDS–PAGE and immunoblotting with the Rab3A rabbit polyclonal antibody.

αGDI–chaperone complex reconstitution

Purified His₆-αGDI was incubated with protein A–Sepharose 4B beads covalently coupled with the αGDI monoclonal antibody to generate αGDI–beads (0.5 µg of His₆-αGDI per 50 µl of beads). αGDI–beads were washed with 0.5 M KCl and 5 mM ATP for 30 min at 4°C to remove unbound protein. Subsequently, αGDI–beads were incubated with 20 µg of purified Hsp90 (Stressgen), 20 µg of purified Hsc70 from bovine brain prepared as described previously (Newmyer and Schmid, 2001) and 2 µg of recombinant Escherichia coli-expressed His₆-CSP (pQE30-CSP vector; a kind gift from Dr R.Burgoyne, University of Liverpool, Liverpool, UK) in buffer A containing 20 mM sodium molybdate and the ATP-regenerating system. In the presence of AMP-PNP, an ATP-regenerating system with AMP-PNP instead of ATP was used. The assay mixtures were incubated for 30 min at 30°C with shaking. At the end of the incubation, the pellets were washed three times with buffer A containing 20 mM sodium molybdate. Complex formation was examined by SDS–PAGE followed by Coomassie Blue staining. Where indicated, GA was added at the indicated concentration and incubated for 10 min at 30°C prior to the addition of αGDI–beads.

Mass spectrometry

Protein bands were excised from the colloidal Coomassie Blue (Sigma)-stained SDS–PAGE gel and digested in-gel with trypsin and analyzed by the TSRI Mass Spectroscopy Core Facility.