Association of l-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles
| Autor: | Kathleen Davis, Todd Foos, Erik Floor, John V. Schloss, Chao-Yuh Yang, John B. Tyburski, Jang-Yen Wu, Hong Jin, Che-Chang Hsu, Weiqing Chen |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
endocrine system
animal structures Immunoprecipitation Glutamate decarboxylase Neurotransmission Biology Biochemistry Synaptic vesicle Heat shock protein Animals Humans HSP70 Heat-Shock Proteins Molecular Biology Integral membrane protein gamma-Aminobutyric Acid chemistry.chemical_classification Neurotransmitter Agents Glutamate Decarboxylase HSC70 Heat-Shock Proteins Antibodies Monoclonal Brain Membrane Proteins Cell Biology HSP40 Heat-Shock Proteins Precipitin Tests Recombinant Proteins Hsp70 Amino acid Enzyme Activation chemistry Cattle Synaptic Vesicles Carrier Proteins |
| Zdroj: | Journal of Biological Chemistry. 275:20822-20828 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m001403200 |
| Popis: | Recently we have reported that the membrane-associated form of the gamma-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 gamma-aminobutyric acid neurotransmission. |
| Databáze: | OpenAIRE |
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