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

Abstract

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 GAD 65 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 GAD 65 -specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD 65 and GAD 67 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.L-Glutamate decarboxylase (GAD 1 ; EC 4.1.1.15) is the ratelimiting 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 GAD 65 and GAD 67 , referring to GAD with molecular masses of 65 and 67 kDa, respectively (for review, see Ref.2). GAD 67 is mostly soluble and is distributed evenly throughout the cell, whereas GAD 65 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 Ca 2ϩ -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 GAD 65 and/or GAD 67 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 se...

Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

“…5d, lane 3). These results suggest that both HSC70 and CSP form a protein complex with GAD 65 in GABAergic SV, confirming our previous findings in synaptosomal preparation (15).…”

“…Hence, it is clear that two separate protein entities are responsible for synthesis and packaging of GABA into SVs. Recently, we have shown that GAD 65 may become anchored to SVs through protein complex formation first with HSC70, followed by interaction with CSP, an intrinsic SV protein (15). In this article, we have shown the presence of HSC70 and CSP in purified GABAergic SV (Fig.…”

“…Previously, we reported that MGAD is activated by phosphorylation that requires an electrochemical gradient across the SV membrane (7). A model for the anchoring mechanism of GAD to SV and its role as a link between GABA synthesis and storage in nerve terminals was also proposed (15). The evidence presented here will demonstrate that GABA synthesized by SV-associated GAD is preferentially transported into the SV by vesicular GABA transporters (VGATs).…”

“…Subpopulations of GAD 65 and GAD 67 remain firmly anchored to membranes despite various ionic extraction methods (2,4,8). The interaction of GAD with membranes was reported to be through ionic (9)(10)(11), hydrophobic (12,13), protein phosphorylation (14), or proteinprotein interaction (15). Previously, we reported that MGAD is activated by phosphorylation that requires an electrochemical gradient across the SV membrane (7).…”

A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

“…This suggests that GVG does not alter GABA transport into synaptic vesicles. Future studies of this protein complex, which in addition to GAD and VGAT includes heat shock protein 70, cysteine string protein, and CaMKII (Hsu et al, 2000;Jin et al, 2003), might elucidate the relationship between GABA synthesis and transport into synaptic vesicles.…”