Association of L-glutamic acid decarboxylase to the 70-kDa heat shock protein as a potential anchoring mechanism to synaptic vesicles

Recently we have reported that the membrane-associated form of the gamma-am inobutyric acid-synthesizing enzyme, L-glutamate decarboxylase (MGAD), is r egulated by the vesicular proton gradient (Hsu, C. C,, Thomas, C., Chen, W. , Davis, K. M., Foes, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., a nd Wu, J. Y. (1999) J. Biol. Chem. 274, 24366-24371). In this report, sever al lines of evidence are presented to indicate that L-glutamate decarboxyla se (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 76 (HSC70), followed by interaction with cysteine string prot ein (CSP), an integral protein of the synaptic vesicle. The first Line of e vidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution stud ies, HSC70 was found to form complex with GAD,, as shown by gel mobility sh ift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipi tation 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 GrAD(65) and GAD(67) are associated with synaptic vesicl es along with HSC70 and CSP. Eased on these findings, a model is proposed t o link anchorage of MGAD to synaptic vesicles in relation to its role in ga mma-aminobutyric acid neurotransmission.